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Keywords:

  • Alzheimer’s disease;
  • amyloid precursor protein;
  • glutamate receptors;
  • NMDA receptors;
  • receptor trafficking

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results and discussion
  5. Concluding comments
  6. Acknowledgements
  7. References
  8. Supporting Information

This is a study of the interaction between the two NMDA neurotransmitter receptor subtypes, NR1/NR2A and NR1/NR2B, and amyloid precursor protein (APP) 695, the major APP variant expressed in neurones. APP695 co-immunoprecipitated with assembled NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors following expression in mammalian cells. Single NR1-1a, NR1-2a, NR1-4bc-Myc, or NR2 subunit transfections revealed that co-association of APP695 with assembled NMDA receptors was mediated via the NR1 subunit; it was independent of the NR1 C1, C2, and C2′ cassettes and, the use of an NR1-2ac-Myc-trafficking mutant suggested that interaction between the two proteins occurs in the endoplasmic reticulum. The use of antibodies directed against extracellular and intracellular NR2 subunit epitopes for immunoprecipitations suggested that APP/NMDA receptor association was mediated via N-terminal domains. Anti-APP antibodies immunoprecipitated NR1, NR2A, and NR2B immunoreactive bands from detergent extracts of mammalian brain; reciprocally, anti-NR1 or anti-NR2A antibodies co-immunoprecipitated APP immunoreactivity. Immune pellets from brain were sensitive to endoglycosidase H suggesting that, as for heterologous expression, APP and NMDA receptor association occurs in the endoplasmic reticulum. Co-expression of APP695 in mammalian cells resulted in enhanced cell surface expression of both NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors with no increase in total subunit expression. These findings are further evidence for a role of APP in intracellular trafficking mechanisms. Further, they provide a link between two major brain proteins that have both been implicated in Alzheimer’s disease.

Abbreviations used:
ApoEr2

apolipoprotein E receptor 2

APP

amyloid precursor protein

amyloid-β peptides

HEK

human embryonic kidney cells

LTP

long-term potentiation

NR1, NR2A etc.

NMDA receptor NR1 subunit, NR2A subunit etc.

PSD

post-synaptic density

SAP

synapse associated protein

Amyloid precursor protein (APP) is a ubiquitously expressed type I transmembrane protein that undergoes regulated proteolytic processing by secretase enzymes to yield various proteolytic fragments. There are two proteolytic pathways, the non-amyloidogenic and the amyloidogenic pathway mediated via α- and γ-secretases and via β- and γ-secretases, respectively. The latter processing pathway results in the generation of neurotoxic amyloid-β peptides (Aβ) that aggregate to form extracellular amyloid plaques, one of the key pathological traits of Alzheimer’s disease. APP has therefore been the subject of intense investigation. Despite all this activity, however, its functional role remains elusive. Most evidence suggests that APP has a trophic function promoting neurite outgrowth, neuronal migration and repair via interaction with extracellular matrix proteins but definitive proof and universal acceptance of this ascribed role has yet to be obtained (reviewed in Wolfe and Guenette 2007; Suzuki and Nakaya 2008; Thinakaran and Koo 2008; Goedert and Spillantini 2006).

One of the early neurochemical changes in Alzheimer’s disease is dysfunction of cholinergic and glutamatergic synapses. The pervading view is that these changes are marked by synaptic loss followed in the later stages of the disease by neuronal cell death (reviewed in Selkoe 2002). NMDA receptors are one of the major mediators of glutamatergic neurotransmission. They are central to a plethora of physiological and pathophysiological processes in the brain. They are probably best known for their roles in long-term potentiation (LTP) and long-term depression, thought to be molecular mechanisms of learning and memory and, in the initiation of neurodegenerative processes. NMDA receptor channels are highly permeable to calcium ions thus uncontrolled activation leads to an unregulated influx of calcium ions resulting in neuronal cell death (reviewed in Waxman and Lynch 2005).

There are several links between APP and NMDA receptor biology. For example, in cerebrocortical cultures, NMDA receptor activation increased Aβ production and secretion via inhibition of α-secretase candidate tumor necrosis factor-α converting enzyme (Lesnéet al. 2005). Aβ peptides were shown to reduce LTP but facilitate long-term depression (Kim et al. 2001) and also, to affect the trafficking of cell surface NMDA receptors (Snyder et al. 2005). The apolipoprotein E receptor 2 (ApoEr2) was shown to co-immunoprecipitate with APP (Hoe et al. 2005). In a subsequent study, ApoEr2 co-immunoprecipitated with NMDA receptors and the NMDA receptor associated scaffold protein, post-synaptic density-95 (PSD-95), thereby suggesting a possible association between APP and NMDA receptors mediated via ApoEr2 and/or PSD-95 (Hoe et al. 2006a). Disabled 1 (Dab1) is an adaptor protein that associates with APP and also, with ApoEr2. The extracellular matrix protein, Reelin, increased the association between APP and Disabled 1 and in parallel, decreased the processing of APP and ApoEr2 (Hoe et al. 2006b). Reelin was recently shown to regulate the surface trafficking of NMDA receptors (Groc et al. 2007). Further, synapse associated protein 97 (SAP97), another member of the PSD-95 family of scaffolds that also associates with NMDA receptors, was shown to associate with ADAM10 (Kuzbanian), the most accredited candidate for α-secretase (Marcello et al. 2007). Perturbation of ADAM10/SAP97 association was found to result in a decrease in APP processing (Marcello et al. 2007). Finally, two recent reports demonstrated that APP is present in the PSD of cultured neurones where it partially co-distributed with PSD-95 and NMDA receptor subunits (NR1) (Hoe et al. 2009; Hoey et al. 2009). Hoe et al. (2009) further showed that APP770 interacted with NMDA receptors resulting in an increase in the surface expression of the NR2B-containing receptor subtype. This led to the conclusion that APP has a physiological role in enhancing NMDA receptor function. Here, we report new insights into the molecular interactions between APP695, the APP splice form expressed in neurones and NMDA receptors.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results and discussion
  5. Concluding comments
  6. Acknowledgements
  7. References
  8. Supporting Information

Constructs and antibodies

The constructs, pCISNR1-1a, pCISNR1-2a, pCISNR1-2ac-Myc, pCISNR2A, pCISNR2B, pCISNR2BFLAG, and pCI-neoAPP695 were as previously described (Cik et al. 1993; Rutter et al. 2002; Hawkins et al. 1999; Perkinton et al. 2004). pGW1PSD95αc-Myc and pCMVneoSAP102c-Myc were kind gifts of Dr M. Sheng (Massachusetts Institute of Technology, Boston, MA, USA). pCISNR1-4bc-Myc was generated by insertional mutagenesis with the c-Myc epitope inserted between amino acids 898 and 899 of the immature sequence. pCI-neoAPP695FLAG was generated by insertional mutagenesis using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and the nucleotide sequence of the construct was verified by nucleotide sequencing (MWG Biotech AG, Ebersberg, Germany). The oligonucleotide primers used were: forward primer, 5′-GGAGGTACCCACTGAT TACAAGGATGACGACGATAAGGGTAATGCTGGCCTGC-3′; reverse primer, 5′-GCAGGCCAGCATTACCCTTATCGTCGTCAT CCTTGTAATCAGTGGGTACCTCC-3′ to insert the FLAG epitope (codons underlined) between D6 and G7 of the mature APP695 sequence. Anti-NR1 C2; anti-NR2A (44–58), anti-NR2A (1381–1394); anti-NR2B (46–60); and anti-NR2A/2B (raised against the amino acid sequence NR2A 1435–1445 but recognizes both NR2A and NR2B) NMDA receptor antibodies were raised, generated, and affinity-purified as previously described (Chazot et al. 1992; Hawkins et al. 1999; Groc et al. 2006). Anti-APP polyclonal antibodies were raised in rabbits against the APP C-terminal sequence with an additional N-terminal cysteine, i.e., C-NGYENPTYKFFEQMQN, coupled via the cysteine to keyhole limpet hemocyanin. This sequence corresponded to amino acids 679–695 of APP695. The anti-APP695 (679–695) antibodies were affinity-purified using the peptide as the ligand. All anti-APP695 (679–695) affinity-antibody production was carried out by Eurogentec Ltd. (Southampton, Hants, UK). Anti-FLAG M2 antibodies were from Sigma Aldrich (Poole, Dorset, UK); anti-APP (44–62) antibodies were from Chemicon (Temecula, CA, USA); anti-NMDA receptor NR2B antibodies were from Millipore (Massachusetts, MA, USA) anti-c-Myc clone 4A6 antibodies were from Upstate USA Inc. (Charlottesville, VA, USA). The specificity of these anti-NR2B antibodies is shown in the Supporting Information Fig. S1.

Mammalian cell transfection

Human embryonic kidney cells (HEK293) were cultured and transfected with either pCISNR1-1a; pCISNR1-2a; pCISNR1-4bc-Myc; pCISNR2A; pCISNR1-1a/pCISNR2A; pCISNR1-1a/pCISNR2B; pCISNR1-2a/pCISNR2A; or pCISNR1-2ac-Myc/pCISNR2A in the presence and absence of pCI-neoAPP695, pCI-neoAPP695FLAG, pGW1PSD95αc-Myc, or pCMVneoSAP102c-Myc using the calcium phosphate method. For single NMDA receptor subunit and APP695 transfections, a total of 10 μg DNA was used with a ratio of 1 : 1. For NR1 : NR2 binary combinations, a total of 20 μg DNA were used. NMDA receptor and either APP695, PSD-95, or SAP102 were co-transfected using a 1 : 1 ratio; NR1 : NR2 binary combinations used a 1 : 3 ratio thus a triple transfection used, e.g., 2.5 μg pCISNR1-1a : 7.5 μg pCISNR2A : 10 μg pCI-neoAPP695. In the absence of APP695, PSD-95, or SAP102, the pCIS empty vector was included in the transfection mix. Post-transfection cells were cultured in the presence of 1 mM ketamine to prevent NMDA receptor-mediated cytotoxicity. Cells were harvested 24–36 h post-transfection and analyzed by quantitative immunoblotting or were used for immunoprecipitation assays. For transfections where cell surface NMDA receptor expression was measured, HEK293 cells were subcultured overnight prior to transfection in poly-d-lysine (100 μg/mL)-coated 24-well dishes and 0.5 μg total plasmid DNA was used per well.

Immunoblotting

Immunoblotting was performed as described previously using 25–50 μg of protein/sample precipitated by the chloroform/methanol method and sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions in 7.5% polyacrylamide slab minigels (Papadakis et al. 2004). For all the immunoblots of the immunoprecipitates, for the precipitating, primary antibody, 14% of the immune pellet was applied per gel lane with 42% applied for probing with the two other, different specificity antibodies. Affinity-purified antibodies were used at final concentrations of 1–5 μg/mL; anti-c-Myc Clone 4A6 mouse monoclonal antibodies were used at a dilution of 1 : 1000. Rabbit and mouse horseradish-linked secondary antibodies were used at a final dilution of 1 : 2000 and immunoreactivities were detected using the ECL western blotting system (Amersham Biosciences Ltd., Little Chalfont, Bucks., UK). Immunoreactive bands were quantified using the GeneGnome Chemiluminescence Capture and Analysis System (Syngene, Cambridge, UK). For the immunoblots of brain tissue, the transfer was to polyvinylidene difluoride membranes, a dilution of 1 : 10 000 of anti-rabbit horseradish-linked secondary antibodies was used and the blots were developed using the SuperSignal® West Femto ECL substrate (Perbio Science UK Ltd., Northumberland, UK).

Immunoprecipitation assays

Transfected HEK293 cells were harvested 24 h post-transfection, cell homogenates prepared and solubilized for 1 h at 4°C with 50 mM Tris–citrate, pH 7.4, 240 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% (v/v) Triton X-100, containing benzamidine (1 μg/mL), bacitracin (1 μg/mL), soybean trypsin inhibitor (1 μg/mL), chicken egg trypsin inhibitor (1 μg/mL), and phenylmethylsulphonyl fluoride (1 mM). Samples were diluted to 1 mg protein/mL and the solubilized material collected by centrifugation at 100 000 g for 40 min at 4°C. Aliquots (800 μL) were incubated with affinity-purified anti-NR1 C2, anti-NR2A (44–58), anti-NR2A (1381–1394) or anti-APP (679–695) antibodies, or protein A purified non-immune Ig (5 μg) overnight at 4°C. Protein A-Sepharose (2.5 mg) was added and samples were incubated for 1 h at 4°C. Immune pellets were collected by centrifugation at 3000 g for 10 s, washed with 3× solubilization buffer as above (3 × 1 mL), solubilized with sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer and analyzed by immunoblotting as described (Papadakis et al. 2004).

For the immunoprecipitations from native tissue, the P2 membrane fraction was prepared from adult rat brain minus the cerebellum, diluted to 1.5 mg protein/mL and solubilized for 1 h at 4°C with 1% (w/v) sodium deoxycholate in 50 mM Tris–HCl, pH 9.0, containing 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, benzamidine (1 μg/mL), bacitracin (1 μg/mL), soybean trypsin inhibitor (1 μg/mL), chicken egg trypsin inhibitor (1 μg/mL), and phenylmethylsulphonyl fluoride (1 mM) as previously described (Chazot and Stephenson 1997a). The detergent soluble fraction was collected by centrifugation at 100 000 g for 40 min at 4°C. It was diluted 1 : 1 with 50 mM Tris–HCl, pH 7.1, containing 240 mM NaCl, 5 mM EGTA, 5 mM EDTA, benzamidine (1 μg/mL), bacitracin (1 μg/mL), soybean trypsin inhibitor (1 μg/mL), chicken egg trypsin inhibitor (1 μg/mL), and phenylmethylsulphonyl fluoride (1 mM) and 1% (v/v) Triton X-100 and used immediately for immunoprecipitation assays as described above except that the test and control precipitating antibodies were increased to 10 μg.

Endoglycosidase H and N-glycosidase F digestion

Anti-NR2A (1381–1394) immune and control anti-Ig pellets from brain were subjected to endoglycosidase H and N-glycosidase F digestion [New England Biolabs (UK) Ltd, Hitchin, Herts, UK] exactly as described (Kenny et al. 2009). Controls were protein samples incubated for 1 h at 37°C in the absence of enzymes. All samples were precipitated using the chloroform/methanol method and analyzed by immunoblotting.

Determination of NMDA receptor cell surface expression by ELISA

The measurement of cell surface NR1/NR2A and NR1/NR2B NMDA receptors was carried out using an ELISA method with affinity-purified antibodies directed against extracellular epitopes of NR2A and NR2B, i.e., anti-NR2A 44–58 Cys (0.125 μg/mL) or anti-NR2B 46–60 Cys (0.5 μg/mL), respectively as described (Papadakis et al. 2004; Cousins et al. 2008). Surface expression was carried out using anti-NR2 antibodies for NR1/NR2A and NR1/NR2B combinations since NR2 subunits are only trafficked to the cell surface when co-expressed with NR1 (McIlhinney et al. 1998).

Results and discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results and discussion
  5. Concluding comments
  6. Acknowledgements
  7. References
  8. Supporting Information

APP695 co-immunoprecipitates with assembled NR1/NR2A NMDA receptors in heterologous expression systems

Immunoprecipitation with anti-NR1 C2 antibodies

There are three major splice variants of APP, APP770, APP751, and APP695. These differ by the presence or absence of the Kunitz protease inhibitor domain and the OX2 domain within the N-terminal region (Fig. 1). HEK293 cells do not have endogenous NMDA receptors but they are known to express the APP751 splice variant (Lammich et al. 1999) but not APP695, the variant that is predominantly expressed in neurones. Thus, in initial immunoprecipitations, HEK293 cells were transfected with NR1-1a/NR2A ± APP695 clones, detergent extracts of the transfected cells prepared, immunoprecipitations carried out using antibodies directed against the intracellular NR1 C-terminus, i.e., anti-NR1 C2 and control, non-immune Ig, and the respective immune pellets were analyzed by immunoblotting. The results are shown in Fig. 1. Anti-NR1 C2 immunoreactive bands (Mr = 120 kDa) were present as expected for the precipitating primary antibody in the immune but not in non-immune pellets. Anti-NR2A (1381–1394), immunoreactive bands (Mr = 180 kDa) were also detected in all immune but not in non-immune pellets demonstrating that NR1-1a subunits are co-assembled with NR2A. Anti-APP immunoreactivity using both N-terminal and C-terminal directed anti-APP antibodies (Mr = 108 kDa ± 5, n = 5; Mr = 107 ± 5 kDa, n = 5, respectively) was also detected in immune but not in non-immune pellets. This was evident for cells transfected with only NR1-1a/NR2A clones and for cells co-expressing APP695 although the APP immune signal was qualitatively stronger in cells expressing exogenous APP695 (Fig. 1b). The use of the different specificity anti-APP antibodies confirmed that anti-NR1 C2 antibodies were co-precipitating full length APP. To corroborate further the co-immunoprecipitation results, an N-terminal FLAG tagged APP695 was co-expressed with NR1-1a/NR2A clones, anti-NR1 C2 immunoprecipitations were carried out but anti-FLAG antibodies were used for the detection of APP695. Anti-FLAG immunoreactivity was detected in immune but not in non-immune pellets (Fig. 1d). The immunoreactive band was coincident with that observed using anti-APP antibodies, i.e., Mr = 114 ± 7 (n = 3). For all the APP immunoblots, it was difficult to distinguish clearly and reproducibly between the different splice forms and glycosylation variants of APP. In some immunoblots, several immunoreactive bands could be clearly resolved whereas in others, a broad band of APP immunoreactivity was observed (see APP immunoreactive bands in Fig. 1b and c). Nevertheless, it can be concluded that anti-NR1 C2 NMDA receptor antibodies co-immunoprecipitate both endogenous and exogenous APP in HEK293 cells. A recent report found that over-expressing APP resulted in aberrant processing of APP (Muresan et al. 2009). But, since the association between APP and NMDA receptors is seen with endogenous albeit the APP751 splice variant showing that co-immunoprecipitation is not dependent on the Kunitz protease inhibitor domain of APP, this suggests that it is physiologically relevant. Further, the co-immunoprecipitations demonstrate the co-association of NR1-1a with APP and NR1-1a with NR2A. This single immunoprecipitation does not, however, determine if all three proteins are together in a single complex, i.e., does APP associate with assembled NR1-1a/NR2A NMDA receptors (see below).

image

Figure 1.  Endogenous APP, exogenous APP695 and APP695FLAG co-associate with assembled NR1-1a/NR2A NMDA receptors in transfected HEK293 cells; demonstration by immunoprecipitation with anti-NR1 C2 antibodies. (a) Schematic diagrams showing the three main splice variants of APP and the specificity of anti-APP antibodies. (b–d) HEK293 cells were transfected with NR1-1a/NR2A clones in the presence and absence of pCI-neoAPP695 or pCI-neoAPP695FLAG. Transfected cell homogenates were harvested, detergent solubilized, the soluble extracts collected by centrifugation at 100 000 g, and either immunoprecipitated with anti-NR1 C2 antibodies or non-immune Ig and immune pellets were analyzed by immunoblotting (b and d) or extracts were analyzed directly by immunoblotting (c), all as described under Experimental Procedures. (b) Cells transfected in the absence and presence of APP695; (c) total cell homogenates transfected with control empty vector (lane 1); APP695 (lane 2) or APP695FLAG and (d) cells co-transfected with APP695FLAG. The gel lane layout for the immunoblots in (b) and (d) is identical where lane 1 = detergent soluble extract; lane 2 = non-immune pellet, and lane 3 = anti-NR1 C2 immune pellet. NR1, NR2A, APPext, and APPint are the antibody specificities used to probe the immunoblots. The symbol ([RIGHTWARDS ARROW]) denotes NR1, NR2A, and APP where appropriate. The positions of molecular weight standards (×103 Da) are shown on the right. The immunoblots are representative of at least n = 3 independent immunoprecipitations from n = 3 independent transfections.

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Reciprocal studies show that APP antibodies co-immunoprecipitate NR1-1a/NR2A NMDA receptors

It was important to demonstrate that antibodies directed against APP could co-immunoprecipitate NMDA receptors thus NR1-1a/NR2A receptors were co-expressed with APP695 in HEK293 cells and immunoprecipitations carried out with either anti-APP (679–695) or non-immune Ig and pellets were analyzed by immunoblotting. In agreement with the studies using antibodies directed against NMDA receptor subunits, APP, NR1-1a, and NR2A were found in the immune but not in control precipitates (Fig. 2).

image

Figure 2.  Reciprocal studies show that APP antibodies co-immunoprecipitate NR1-1a/NR2A NMDA receptors in transfected HEK293 cells. HEK293 cells were co-transfected with NR1-1a/NR2A and APP695 clones, transfected cell homogenates harvested, detergent solubilized, the soluble extracts collected by centrifugation at 100 000 g, immunoprecipitated with anti-APP (679–695) antibodies or non-immune Ig and immune pellets were analyzed by immunoblotting all as described under Experimental Procedures. The gel lane layout is identical for each immunoblot where lane 1 = detergent soluble extract; lane 2 = non-immune pellet and lane 3 = anti-APP (679–695) immune pellet. NR1, NR2A, and APP are the antibodies used to probe the immunoblots. The symbol ([RIGHTWARDS ARROW]) denotes NR1, NR2A, and APP where appropriate. The positions of molecular weight standards (×103 Da) are shown on the right. The immunoblots are representative of at least n = 3 independent immunoprecipitations from n = 3 independent transfections.

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Immunoprecipitation with anti-NR2A and anti-NR2B NMDA receptor antibodies

NR1-1a/NR2A and NR1-1a/NR2BFLAG NMDA receptors were expressed + APP695 in HEK293 cells, detergent extracts of the transfected cells prepared and immunoprecipitations carried out this time using antibodies directed against extracellular or intracellular antigenic determinants of NR2A, i.e., anti-NR2A (44–58) and NR2A (1381–1394) and NR2B subunits, i.e., NR2B (46–60) and NR2A/B (1454–1464), in parallel with control, non-immune Ig. As before, immune pellets were analyzed by immunoblotting. For both antibodies, anti-NR2A immunoreactivity was found in immune but not in non-immune pellets. NR1 subunit immunoreactivity was also found in both immune but not in control, non-immune pellets (Fig. 3). Anti-APP immunoreactivity, however, was only detected in immunoprecipitates where the primary antibody was anti-NR2A (1381–1394). No APP immunoreactivity was detected when immunoprecipitations were carried out with anti-NR2A antibodies directed against an extracellular determinant (Fig. 3b). The same observations were made for NR1-1a/NR2BFLAG combinations. First, as for NR1-1a/NR2A, immunoprecipitation with anti-NR1 C2 antibodies resulted in the detection of NR1, NR2B, and APP in immune but not in control pellets. Second, immunoprecipitation with both specificity anti-NR2B antibodies resulted in detection of NR1-1a and NR2B in pellets [note that anti-NR2A (1454–1464) antibodies that are directed against intracellular epitopes recognizes both NR2A and NR2B]. APP was only detected when antibodies directed against NR2B intracellular determinants were used for the immunoprecipitations (Fig. 3c). As for the immunoprecipitations using anti-NR1 C2 antibodies, these results demonstrate the co-precipitation of NR2A/APP, NR2B/APP and NR1-1a/NR2A, NR1-1a/NR2B combinations, but this single immunoprecipitation does not determine if all three proteins are co-associated in a single complex, i.e., assembled NR1-1a/NR2 NMDA receptors together with APP. They further suggest that since antibodies directed against extracellular epitopes of NR2 subunits do not co-precipitate APP, association between APP and NMDA receptors whether direct or indirect occurs via their respective extracellular N-terminal domains thus precluding primary antibody binding by steric hindrance.

image

Figure 3.  Exogenous APP695 co-associates with assembled NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors in transfected HEK293 cells; demonstration by immunoprecipitation with anti-NR2 antibodies directed against intracellular but not extracellular NR2 epitopes. (a) Schematics of NMDA receptor NR2A and NR2B subunits showing antibody specificities. HEK293 cells were co-transfected with either NR1-1a/NR2A ± APP695 clones (b) or NR1-1a/NR2BFLAG + APP695 clones (c), transfected cell homogenates harvested, detergent solubilized, the soluble extracts collected by centrifugation at 100 000 g, immunoprecipitated with antibodies directed against either an extracellular or an intracellular determinant of NR2A or NR2B subunits or non-immune Ig and immune pellets were analyzed by immunoblotting all as described under Experimental Procedures. The gel lane layout is identical for each immunoblot where lane 1 = detergent soluble extract; lane 2 = non-immune pellet and lane 3 = anti-NR2A or anti-NR2B immune pellet. NR1, NR2A, APP and FLAG are the antibodies used to probe the immunoblots. The symbol ([RIGHTWARDS ARROW]) denotes NR1, NR2A, NR2B, and APP where appropriate. The positions of molecular weight standards (×103 Da) are shown on the right. The immunoblots are representative of at least n = 3 independent immunoprecipitations from n = 3 independent transfections.

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Single subunit expression studies

To resolve the issue of whether APP is interacting with single NMDA receptor subunits or assembled NR1/NR2 complexes, single subunit expression and immunoprecipitations were carried out. When NR1-1a, NR1-2a, and NR1-4bc-Myc splice variants, selected to encompass the full range of NR1 exons, were expressed alone with APP695 and immunoprecipitations carried out with anti-NR1 C2 or anti-c-Myc antibodies, each NR1 splice variant and APP695 were both detected in the immune pellets (Fig. 4b–d). When NR2A was expressed alone with APP695 and immunoprecipitations were carried out with anti-NR2A (1381–1394) antibodies, NR2A immunoreactivity was detected in immune but not in control pellets (Fig. 4e). In contrast to the observations for single subunit NR1-1a expression, no APP695 was detectable in either the immune or the control pellets (Fig. 4e). This suggests that the association of APP695 with NMDA receptors is mediated via interaction of APP695 with the NR1 subunit and it is independent of the N1, C1, C2, and C2′ NR1 cassettes. Furthermore, it shows that APP695 associates with assembled NR1-1a/NR2A receptors since APP695 is found in immune pellets of NR1-1a/NR2A/APP695 transfectants when the precipitating antibody is anti-NR2A (1381–1394) (Fig. 3b). It may be argued that the co-immunoprecipitation of APP with NMDA receptors may be an artifact of heterologous expression since non-physiological, high concentrations of all three proteins may accumulate in the endoplasmic reticulum and be associated non-specifically. However, this would be the case for both NR1-1a and NR2A polypeptides but no co-immunoprecipitation with APP695 is observed for NR2A single subunit expression thus allaying this concern (Fig. 4e).

image

Figure 4.  NR1 but not NR2A NMDA receptor single subunits co-immunoprecipitate with APP695. (a) A schematic showing the cassettes of the NR1 splice variants. HEK293 cells were co-transfected with either the clones, NR1-1a + APP695 (b); NR1-2a + APP695 (c); NR1-4bc-Myc + APP695 (d) or NR2A + APP695 (e), transfected cell homogenates harvested, detergent solubilized, the soluble extracts collected by centrifugation at 100 000 g, immunoprecipitated with anti-NR1 C2 (NR1-1a or NR1-2a + APP695), anti-c-Myc (NR1-4bc-Myc + APP695) or anti-NR2A (1381–1394) (NR2A + APP695) antibodies or non-immune Ig and immune pellets were analyzed by immunoblotting all as described under Experimental Procedures. The gel lane layout is identical for each immunoblot where lane 1 = detergent soluble extract; lane 2 = non-immune pellet and lane 3 = anti-NR1 or NR2A (1381–1394) immune pellet. NR1, NR2A, and APP are the antibodies used to probe the immunoblots. The symbol ([RIGHTWARDS ARROW]) denotes NR1, NR2A, and APP where appropriate. The positions of molecular weight standards (×103 Da) are shown on the right. The immunoblots are representative of at least n = 3 independent immunoprecipitations from n = 3 independent transfections.

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NMDA receptors co-immunoprecipitate with APP in native tissue

Co-immunoprecipitation studies were importantly carried out on 100 000 g detergent extracts of brain tissue. Immunoprecipitations were carried out with either: anti-NR1 C2, anti-NR2A (1381–1394), anti-APP (679–695), or equivalent amounts of control non-immune Ig. In each case, NR1, NR2A, and APP immunoreactivities were all detected in the test but not in control pellets (Fig. 5a–c). For the detection of NR2B subunits, immunoblots of APP in anti-APP immunoprecipitates were reprobed with the commercial anti-NR2B antibody; an immunoreactive band with Mr 180 kDa was clearly evident (Fig. 5c). Thus, in native tissue, APP is co-immunoprecipitated with both NR2A- and NR2B-containing NMDA receptors.

image

Figure 5.  NR2A- and NR2B-containing NMDA receptors co-immunoprecipitate with APP from detergent extracts of adult mammalian brain. Detergent extracts (100 000g) were prepared from adult rat brain, immunoprecipitations carried out using anti-NR1 C2, anti-NR2A (1381–1394), anti-APP (679–695) antibodies or non-immune Ig and immune pellets were analyzed by immunoblotting. (a, b) The immunoprecipitating antibodies are shown above the immunoblots; the gel lane layout is identical for each immunoblot where lane 1 = detergent soluble extract; lane 2 = non-immune pellet and lane 3 = anti-NR1 C2 or NR2A (1381–1394) immune pellet. In (c), the immunoprecipitating antibodies were anti-APP (679–695) and non-immune Ig. Following the development of the immunoblot, it was stripped as under Experimental Procedures and reprobed with anti-NR2B antibodies. NR1, NR2A, and APP are the antibodies used to probe the immunoblots. The symbol ([RIGHTWARDS ARROW]) denotes NR1, NR2A, NR2B, and APP where appropriate. The positions of molecular weight standards (×103 Da) are shown on the right. The immunoblots are representative of at least n = 3 independent immunoprecipitations from at least three separate detergent extract preparations.

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NMDA receptors assemble with APP695 in the endoplasmic reticulum

The immunoprecipitation experiments with anti-NR2 antibodies suggested that APP and assembled NR1/NR2 NMDA receptors associate via their N-terminal domains. APP and NMDA receptors are integral membrane proteins thus they are both trafficked to the plasma membrane along the secretory pathway. In accord with suggestions that APP is involved in intracellular trafficking events (e.g. Kamal et al. 2000), it may be speculated that the two proteins associate at a point from the endoplasmic reticulum onwards and that they remain together as a functional unit following plasma membrane insertion. It may be that they may associate only when both are expressed at the cell surface. Alternatively, it could be that APP on one cell interacts with assembled NR1/NR2A on a second cell in a heterotypic type cell–cell interaction.

To address these possibilities, two experimental strategies were used. For the first, HEK293 cells were transfected in parallel with NR1-1a/NR2A + APP695FLAG, NR1-1a/NR2A, or APP695FLAG clones. Cells were harvested, 100 000g detergent extracts prepared, and the NR1-1a/NR2A and APP695 separate transfectants mixed in a 1 : 1 ratio. Immunoprecipitations were carried out using anti-NR1 C2 and non-immune antibodies as before. From the results shown in Fig. 6, it is seen that in NR1-1a/NR2A + APP695FLAG expressing cells, NR1-1a, NR2A, and APP695 are all detected in the immune but not in the control pellets (Fig. 6a). In the transfectants where NR1-1a/NR2A and APP695 were separately expressed, NR1-1a and NR2A were detected in the immune pellets, however, no FLAG immunoreactivity was detected (Fig. 6b). This finding shows that to co-immunoprecipitate, APP and NR1-1a/NR2A need to be expressed in the same cell suggesting therefore that they do not associate via a heterotypic, cell–cell association.

image

Figure 6.  NMDA receptors and APP695 do not associate via trans cell–cell heterotypic interactions. HEK293 cells transfected in parallel with either with NR1-1a/NR2A + APP695 combined (a) or separately (b) with NR1-1a/NR2A and APP695. Transfected cell homogenates were harvested, detergent solubilized, and soluble extracts collected by centrifugation at 100 000 g. The soluble extracts from the NR1-1a/NR2A and APP695 single transfectants were mixed 1 : 1. Immunoprecipitations with anti-NR1 C2 or non-immune Ig were carried out and immune pellets were analyzed by immunoblotting all as described under Experimental Procedures. The gel lane layout is identical for each immunoblot where lane 1 = detergent soluble extract; lane 2 = non-immune pellet and lane 3 = anti-NR1 C2 immune pellet. NR1, NR2A, and APP are the antibodies used to probe the immunoblots. The symbol ([RIGHTWARDS ARROW]) denotes NR1, NR2A, and APP where appropriate. The positions of molecular weight standards (×103 Da) are shown on the right. The immunoblots are representative of at least n = 3 independent immunoprecipitations from n = 3 independent transfections.

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In the second approach, use was made of an NMDA receptor subunit trafficking mutant, NR1-2ac-Myc, that we previously described (Papadakis et al. 2004). This subunit has a c-Myc epitope tag inserted between amino acids 81/82 of the NR1-2a splice variant. It was shown to behave as wild-type NR1-2a in that it is expressed and it co-assembles with NR2A but, unlike wild-type, when co-expressed with NR2A, the assembled receptors are not trafficked to the cell surface and they remain trapped in the endoplasmic reticulum (Papadakis et al. 2004). Thus, in parallel, HEK293 cells were co-transfected with APP695 + either NR1-1a/NR2A, wild-type NR1-2a/NR2A, or NR1-2ac-Myc/NR2A. Detergent extracts were prepared and immunoprecipitations carried out with anti-NR1 C2, anti-NR2A (1381–1394) or non-immune antibodies and pellets were analyzed by immunoblotting as before. Wild-type NR1-2a/NR2A behaved exactly as found for NR1-1a/NR2A in that NR1, NR2A and APP immunoreactivities were all detected in immune but not in non-immune pellets (Fig. 7b and c) demonstrating as shown earlier for the single subunit expression studies (Fig. 4), that the NR1 C1 cassette is not necessary for the association. Similarly, NR1, NR2A, and APP immunoreactivities were all found in immune but not control non-immune pellets from detergent extracts of cells transfected with NR1-2ac-Myc/NR2A (Fig. 7b and c). This suggests that assembled NMDA receptors associate with APP in the endoplasmic reticulum. It is compatible with the finding that APP associates with the single expressed NR1-1a single subunit since it is known to be retained in the endoplasmic reticulum in the absence of NR2A (McIlhinney et al. 1998).

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Figure 7.  Assembled NR1-1a/NR2A NMDA receptors and APP associate in the endoplasmic reticulum: demonstration using the NR1-2ac-Myc trafficking mutant. (a) Schematic diagrams of the NR1-1a and NR1-2a splice variants. In (b) and (c), HEK293 cells were co-transfected in parallel with the clones, NR1-1a/NR2A + APP695, NR1-2a/NR2A + APP695, and NR1-2ac-Myc/NR2A + APP695. Transfected cell homogenates were harvested, detergent solubilized, the soluble extracts collected by centrifugation at 100 000 g, immunoprecipitated with either anti-NR1 C2 (b) or anti-NR2A (1381–1394) (c) antibodies or non-immune Ig and immune pellets were analyzed by immunoblotting all as described under Experimental Procedures. The gel lane layout is identical for each immunoblot where lane 1 = detergent soluble extract; lane 2 = non-immune pellet and lane 3 = anti-NR1 C2 or NR2A (1381–1394) immune pellet. NR1, NR2A, and APP are the antibodies used to probe the immunoblots. The symbol ([RIGHTWARDS ARROW]) denotes NR1, NR2A, and APP where appropriate. The positions of molecular weight standards (×103 Da) are shown on the right. The immunoblots are representative of at least n = 3 independent immunoprecipitations from n = 3 independent transfections.

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To determine if association between APP and NMDA receptors also occurred in the endoplasmic reticulum in native brain tissue, use was made of the sensitivity of both proteins to endoglycosidase H digestion. Assembled NR1/NR2A receptors and APP are both trafficked to the plasma membrane via the secretory pathway; both also have consensus amino acid sequences for N-glycosylation. In the endoplasmic reticulum, proteins destined for secretion are post-translationally modified by the addition of high mannose residues to appropriate asparagines. These can be removed by the glycolytic enzyme, endoglycosidase H. The high mannose residues are trimmed back as the proteins process through the Golgi apparatus thus N-linked glycoproteins become resistant to endoglycosidase H digestion. Sensitivity to endoglycosidase H digestion is therefore a diagnostic marker for endoplasmic reticulum localization. Thus, anti-NR2A (1381–1394) immune and non-immune pellets were subjected to endoglycosidase H and also N-glycanase F treatment together with appropriate temperature controls. (N-glycosidase F cleaves N-glycoproteins at the asparagine where N-linked carbohydrate modification occurs thus it acts as a ‘positive’ control and is indicative of mature post-endoplasmic reticulum proteins). Samples were analyzed by immunoblotting (Fig. 8). For NR1 and NR2 subunits, it was evident that there was a decrease in their respective molecular weights following N-glycanase F digestion. For both, a single immunoreactive species with a decrease in Mr = 10 kDa (NR1) and 8 kDa (NR2A) was found. A decrease in the molecular weights was also evident for NR1 and NR2A following endoglycosidase H treatment. For NR1 and NR2A, again only a single immunoreactive band with a decreased Mr coincident with the N-glycanase band was generally observed, i.e., an endoglycosidase H-sensitive fraction. This would be representative of endoplasmic reticulum located receptor. In some immunoblots, an upper band was also detectable indicating an insensitive fraction which would correspond to post-endoplasmic reticulum proteins. However, this was always the minor band suggesting that the majority of the NMDA receptors in the immune pellet were resident in the endoplasmic reticulum. This may be explained by the difficulty of solubilizing synaptic NMDA receptors due to their association with the PSD hence detergent extracts of brain are enriched in intracellular NMDA receptors. For APP, it was more difficult to resolve the immunoreactive bands because of the multiple glycosylated forms of APP. However, it was evident that treatment with endoglycosidase H resulted in an enrichment of the lower molecular weight species (Fig. 8) suggesting that there is co-association between NMDA receptors and APP in the endoplasmic reticulum and implying that, like in heterologous cells, the two proteins interact early in the secretory pathway.

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Figure 8.  Endoglycosidase H sensitivity demonstrates that NR2A-containing NMDA receptors associate with APP in native tissue in the endoplasmic reticulum. Detergent extracts (100 000g) were prepared from adult rat brain, immunoprecipitations carried out using anti-NR2A (1381–1394) or non-immune Ig. Immune and non-immune pellets were collected by centrifugation, resuspended, incubated at 37°C in the presence and absence of deglycosylating enzymes as shown and then analyzed by immunoblotting using either anti-NR1, NR2A, or APP antibodies again, as shown, all as described in Experimental Procedures. Lane 1 = non-immune pellet; lane 2 = anti-NR2A (1381–1394) immune pellet. The results are representative of four immunoblots from four independent immunoprecipitations from four separate detergent extract preparations.

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APP695 enhances surface expression of NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors

Hoe et al. (2009) reported that siRNA knock-down of APP reduced NMDA receptor currents in cerebellar granule and hippocampal neurones. Conversely, they found that over-expression of APP770 in both cell types resulted in enhanced NMDA receptor currents which was attributed to an increase in the cell surface NR2B-containing receptors. Antibody feeding studies revealed that this increase was because of a decrease in NR2B-containing receptor internalization. Here, we investigated the effect of APP695 on the surface trafficking of both NR2A- and NR2B-containing receptors in mammalian cells using positive and negative controls. The positive control was transfection of NR1-1a/NR2A and NR1-1a/NR2B + the scaffold protein, PSD-95, a member of the membrane associated guanylate kinase family of proteins. We previously reported that PSD-95 enhances NR1/NR2A and NR1/NR2B NMDA receptor surface expression. This is a result of a selective increase in NR2A or NR2B total subunit expression. The increased NR2 subunits assemble with the pool of unassembled NR1 subunits to result in an enhancement in surface receptor number (Rutter et al. 2002; Cousins et al. 2008). The negative control was transfection of NR1-1a/NR2A and NR1-1a/NR2B + the scaffold protein, SAP102, also a membrane associated guanylate kinase family member. SAP102 has no effect on NMDA receptor surface expression (Cousins et al. 2008). Figure 9 shows the results. APP695 enhanced the surface expression of both receptor subtypes. Fold increases in surface expression induced by APP695 were: NR1-1a/NR2A, 1.7 ± 0.1 (n = 14) and NR1-1a/NR2B, 2.1 ± 0.2 (n = 8). These compared with fold increases induced by PSD-95 and SAP102, respectively for NR1-1a/NR2A 2.0 ± 0.2 (n = 14); 1.0 ± 0.1 (n = 14) and for NR1-1a/NR2B, 1.9 ± 0.2 (n = 8); 1.1 ± 0.1 (n = 8) (Fig. 9a and b). In contrast to PSD-95 where the increase in surface expression was a result of a total increase in NR2 subunit expression, APP695 had no effect on the level of neither NR1-1a nor NR2 total expression. Values for the fold increases were: NR1-1a, 1.2 ± 0.2, NR2A, 1.0 ± 0.1 (n = 3) for NR1-1/NR2A expression; and NR1-1a, 1.0 ± 0.1, NR2B, 1.0 ± 0.1 (n = 3) for NR1-1a/NR2B (Fig. 9c and d).

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Figure 9.  APP695 enhances surface expression of NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors. HEK293 cells were co-transfected in triplicate with either: NR1-1a/NR2A; NR1-1a/NR2A + PSD-95; NR1-1a/NR2A + SAP102; NR1-1a/NR2A + APP695; NR1-1a/NR2B; NR1-1a/NR2B + PSD-95; NR1-1a/NR2B + SAP102; NR1-1a/NR2B + APP695 clones. Cell surface receptor expression was measured 20 h post-transfection using anti-NR2A 44–58 Cys or anti-NR2B 46–60 Cys antibodies as appropriate (a, b) or, cell homogenates were collected and analyzed by quantitative immunoblotting (c, d) all as described in Experimental Procedures. In (a) and (b), the results were expressed as the fold increase in cell surface expression where the expression of control transfections, i.e., NR1-1a/NR2A/pCIS = 1. Values are the means ± SEM for n = 14 (NR1-1a/NR2A) and n = 8 NR1-1a/NR2B independent transfections. In (c) and (d), the results are expressed as the fold change in subunit expression, i.e., in the presence divided by in the absence the absence of exogenous APP695. Results are the mean ± SEM for n = 3 immunoblots from three independent transfections. ***< 0.001.

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Hoe et al. (2009) showed that the enhanced surface expression of NR2B-containing NMDA receptors was because of a decrease in receptor internalization. This suggests that APP interacts with this receptor subtype in the membrane at the neuronal cell surface. However, it was not established whether the interaction between the two proteins is direct or mediated via an intermediary protein. The results that are reported herein differ from (Hoe et al. 2009) in that enhanced cell surface expression of both NR2A- and NR2B-containing receptors is elicited by APP695 although admittedly, this is for heterologous expression of recombinant receptors. They also differ in that association occurs early in the secretory pathway, within the endoplasmic reticulum, suggesting a regulatory role on forward trafficking of receptors. Further, there is the conundrum that in the ELISA assays for the measurement of steady state cell surface receptor expression, antibodies directed against extracellular epitopes of the NR2A and NR2B subunits were used yet these antibodies did not co-immunoprecipitate NMDA receptors and APP695. A possible explanation for this was the inability of the antibodies to access NR2 extracellular epitopes because of steric hindrance by APP695. This suggests that at the cell surface, this putative steric hindrance is not a factor since the two proteins are no longer associated. Alternatively, it may reflect the fact that in the immunoprecipitations, the receptors are in their native conformation whereas the cell surface ELISA assays are carried out on fixed cells. The presentation of the epitope may therefore be different between the two paradigms resulting in antibody binding to extracellular NR2 epitopes in fixed transfected cells.

Concluding comments

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results and discussion
  5. Concluding comments
  6. Acknowledgements
  7. References
  8. Supporting Information

Multiple protein–protein interactions are known to contribute toward the delivery of NMDA receptors to targeted sites within synaptic membranes and also, to the expression of appropriate receptor numbers at the cell surface. A recent report identified a new NMDA receptor interacting protein, APP770, thus uncovering a link between these two brain proteins both of which have been independently associated with neurodegenerative disease (Hoe et al. 2009). The results reported here concur in part with Hoe et al. (2009). We show here that APP695, the neuronal splice variant, associates with assembled NR1/NR2 NMDA receptors via the NR1 subunit, that the experimental findings are consistent with this association being mediated via the NR1 and APP695 N-terminal domains and that this association occurs early in the secretory pathway in the endoplasmic reticulum. Further, we report that both NR2A- and NR2B-containing receptors can associate with APP695. This is in contrast to results reported by Hoe et al. (2009) where functional measurements using NMDA receptor subtype-selective antagonists showed that association in neurons was with NR2B-containing receptors only. Anti-APP and anti-NR1 immunoprecipitates were not probed with anti-NR2 antibodies. A further consideration is that NR2A and NR2B subunits are known to co-exist in NR1/NR2 assembled native receptors (Chazot and Stephenson 1997b; Al-Hallaq et al. 2007). It may be that the pharmacological tools employed for the functional studies are not sufficient to discriminate between NR1/NR2B and NR1/NR2A/NR2B receptors.

Overall, these results give support to the function of APP as a regulator of intracellular trafficking mechanisms. It may be speculated that APP could be an NMDA receptor auxiliary subunit analogous to the transmembrane α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor regulatory proteins (Tomita et al. 2003) or Clr/Cls, Uegf, Bmp1 (CUB) domain protein neuropilin tollid-like 1 (Neto1), a recently described putative NMDA receptor ancillary protein (Ng et al. 2009). Further work is needed to establish if the association between APP and assembled NMDA receptors is direct or whether it is mediated via an intermediary protein, and also, if this association is maintained on the cell surface. Interestingly, mice that lack APP were shown to develop age-dependent deficits in cognitive function (Dawson et al. 1999), loss of synaptic marker immunoreactivities in the hippocampus and in the cerebral cortex implicating synaptic loss (Dawson et al. 1999) and impairments in LTP (Dawson et al. 1999; Seabrook et al. 1999). Further, a more recent report demonstrated a link between synaptic NMDA receptor activity and APP processing (Hoey et al. 2009) suggesting a high order of molecular organization within the post-synaptic membrane which may contribute to normal physiological functions such as synaptic plasticity. Understanding these basic processes may lead to new insights into the causes of Alzheimer’s disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results and discussion
  5. Concluding comments
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Professors S. Nakanishi and M. Mishina for the gifts of the original NMDA receptor clones, Professor M. Sheng for the gifts of pGW1PSD95αc-Myc and pCMVneoSAP102c-Myc, Professor Chris C.J. Miller (Institute of Psychiatry, King’s College London, UK) for the gift of pCI-neoAPP695, Dr Lynda Hawkins for generating pCISNR1-4bc-Myc and Dr Kieran Brickley, School of Pharmacy for help with antibody production. This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), UK, The Alzheimer’s Society, UK and The Alzheimer’s Research Trust, UK.

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  2. Abstract
  3. Experimental procedures
  4. Results and discussion
  5. Concluding comments
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results and discussion
  5. Concluding comments
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. Demonstration of the specificity of the commercial anti-NR2B antibodies.

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