Differential Regulation of N-Methyl-D-Aspartate Receptor Subunits is An Early Event in the Actions of Soluble Amyloid-β 1-40 Oligomers on Hippocampal Neurons

Abstract

Synaptic dysfunction during early stages of Alzheimer’s disease (AD) is triggered by soluble amyloid-β (Aβ) oligomers that interact with NMDA receptors (NMDARs). We previously showed that Aβ induces synaptic protein loss through NMDARs, albeit through undefined mechanisms. Accordingly, we here examined the contribution of individual NMDAR subunits to synaptotoxicity and demonstrate that Aβ exerts differential effects on the levels and distribution of GluN2A and GluN2B subunits of NMDAR in dendrites. Treatment of cultured hippocampal neurons with Aβ_1-40 (10 μM, 1 h) induced a significant increase of dendritic and synaptic GluN2B puncta densities with parallel decreases in the puncta densities of denritic and synaptic pTyr¹⁴⁷²-GluN2B. Conversely, Aβ significantly decreased dendritic and synaptic GluN2A and dendritic pTyr¹³²⁵-GluN2A puncta densities and increased synaptic pTyr¹³²⁵-GluN2A puncta densities. Unexpectedly, Aβ treatment resulted in a significant reduction of GluN2B and pTyr¹⁴⁷²-GluN2B protein levels but did not influence GluN2A and pTyr¹³²⁵-GluN2A levels. These results show that Aβ exerts complex and distinct regulatory effects on the trafficking and phosphorylation of GluN2A and GluN2B, as well as on their localization within synaptic and non-synaptic sites. Increased understanding of these early events in Aβ-induced synaptic dysfunction is likely to be important for the development of timely preventive and therapeutic interventions.

Keywords

Alzheimer’s disease amyloid beta hippocampus NMDA receptor subunits synapse tyrosine phosphorylation

INTRODUCTION

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder, characterized by a gradual loss of memory and other cognitive functions. Increasingly, evidence suggests that the cognitive decline observed in AD subjects reflects subtle synapticdysfunction rather than neuronal loss per se [1, 2]. Early synaptic dysfunction in AD has been attributed to soluble Aβ oligomers [3, 4] acting in a glutamate-dependent manner [5 –8]. The main species of Aβ found in amyloid deposits are Aβ_1-40 and Aβ_1-42 [9]. In human brain, the most abundant peptide is Aβ_1-40 [10 –12], which has been implicated in synaptic loss in AD patients [13 –15]. Soluble Aβ oligomers are thought to exert synaptotoxic effects by binding near, or to, ionotropic N-methyl-D-aspartate receptors (NMDARs) [16], which play an essential role in mediating excitatory neural transmission, synaptic plasticity and glutamate-induced excitotoxicity by allowing the passage of Ca^2 + into neurons.

NMDARs exist as heteromeric complexes, and include two obligatory GluN1 and two modulatory GluN2 (GluN2A and GluN2B) subunits; subunit composition determines NMDAR sensitivity and electrophysiological properties [17, 18], as well as Aβ-induced neurotoxicity [19]. The composition of synaptic NMDAR subunits changes during development, switching from predominantly GluN2B to GluN2A receptors [20]. The GluN2A subunit is primarily found at synaptic sites and is characterized by rapid offset kinetics, while the GluN2B subunit is preferentially located at extrasynaptic sites and displays slower kinetics [21 –23]. Many recent studies have demonstrated that NMDARs can exchange and diffuse laterally between synaptic and non-synaptic sites [24] under the modulatory influence of various factors, including scaffold proteins (e.g., PSD-95, SAP102) [25]. Dysregulated NMDAR subunit trafficking has been suggested as a mechanism that contributes to synaptic dysfunction in AD [26]. Importantly, post-translational modifications of NMDARs, such as tyrosine phosphorylation, are known to regulate NMDAR expression [26, 27]and function [26, 28]. To date, only GluN2 subunits have been demonstrated to be subject to tyrosine phosphorylation [29]; although phosphorylation of the Tyr 1325 and Tyr 1472 sites within the GluN2A and GluN2B subunits, respectively, are considered to be crucial events in NMDAR activation [30, 31], there is still little clarity regarding the precise role of each Tyr-phosphorylated GluN2 subunit in the execution of Aβ-induced synaptotoxicity [26 , 30–32].

Here, we demonstrate the differential regulation of GluN2A and GluN2B, the phosphorylation of that GluN2A^Tyr1325 and GluN2B^Tyr1472 by Aβ_1-40 at synaptic sites of hippocampal neurons; Aβ also downregulated the total levels of GluN2B/ GluN2B^Tyr1472/Fyn proteins. Importantly, these changes were observed after just 1 h exposure to Aβ.

MATERIALS AND METHODS

Chemicals and reagents

Aβ_1–40 (Cat.#A1075), bovine serum albumin (BSA, Cat.# A7030), sodium borate, gelatin, poly-D-lysine (PDL), and trypsin inhibitor were obtained from Sigma. Antibodies to the following proteins were purchased from: Sigma (rabbit anti-phospho-NMDA NR2B(pTyr¹⁴⁷²), Cat.#M2442; rabbit anti-SAP102, Cat.#S4687; mouse anti-β-actin, Cat.#A1978), Millipore (rabbit anti-NR1, Cat.#AB9864; rabbit anti-NR2A, Cat.#AB1555P; rabbit anti-NR2B, Cat.#AB1557P), BD Biosciences Pharmingen (mouse anti-synapsinI, Cat.#611392), Synaptic Systems (rabbit anti-SAP102, Cat.#124213). Rabbit anti-NMDAR2A (phospho Y¹³²⁵) (Cat.# ab16646 and Cat.#ab106590) were from Abcam. Rabbit anti-Fyn (Cat.#4023) and mouse anti-Src (L4A1) (Cat.#2110) were from Cell Signaling. Anti-mouse and anti-rabbit sera, conjugated to horseradish peroxidase (HRP) were obtained from Bio-Rad. Alexa Fluor^® 594-conjugated goat anti-mouse secondary antibody and Alexa Fluor^® 488-conjugated goat anti-rabbit secondary antibody were purchased from Invitrogen.

Primary hippocampal neurons culture

Primary cultures of rat hippocampal cells were prepared from postnatal day 4 (P4) Wistar rats as described previously [15 , 34]. Trypsin dissociated cells were plated at a density of 450,000 cells/mm² on gelatin/PDL coated glass coverslips. Cultures were maintained in Neurobasal medium supplemented with B27, containing Glutamax I, kanamycin and basic fibroblast growth factor (bFGF) (all materials from Invitrogen). Cells maintained in a humidified incubator (95% air, 5% CO₂) at 37°C. Half of the medium was replenished every 3 days during the culture period. Cells were treated with Aβ and monitored after 7 days in vitro (DIV 7).

Soluble Aβ_1-40 oligomers preparation and treatment

Soluble Aβ_1-40 oligomers were prepared as described previously [15 , 36]. Briefly, Aβ_1-40 peptide was dissolved in 2 mM DMSO, diluted in PBS, vortexed for 30 min, and centrifuged (1 h, 15,000 g, 4°C). The predominant aggregates in such preparations are reported to be low N-oligomers (mainly monomeric to tetrameric) [9 , 37–39]. It should be noted thatfibrillogenesis requires longer incubation times and higher concentrations (>10 μM) of the peptide [40, 41]. In our present experiment, the oligomer nature of Aβ_1-40 in such preparation was also confirmed with atomic force microscopy (AFM) (Fig. 1).

The chosen dose (10 μm) and duration of exposure (1 h) to Aβ in this study was based on our previous observation that these parameters all detection of early synaptotoxicity [33].

Immunofluorescence

Cells were fixed in ice cold 4% paraformaldehyde, washed, permeabilized (0.3% Triton X-100 in PBS) and blocked. Specimens were incubated (overnight, 4°C) with primary antibodies against GluN1 (1:1000), GluN2A (1:1000), GluN2B (1:1000), Synapsin I (1:4000), SAP102 (1:1000), GluN2A/Tyr¹³²⁵ (1:200), and GluN2B/pTyr¹⁴⁷² (1:500). Immunoreactive antigens were visualized after staining with Alexa Fluor^® 594- conjugated goat anti-mouse IgG (1:500) or Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1:500). Nuclear staining was achieved using Hoechst 33342 (1:1000). Optical sections and stacks of images from fluorescence labeled cells were captured with a Leica confocal laser scanning microscope (TCS SP5, with a plan apochromat 63×/1.4 numerical aperture oil lens) or a Leica fluorescence microscope (DM5000).

Puncta analysis

For quantification, 5–10 neurons from 5 different batches of cultures and experiments were randomly chosen for evaluating puncta density (L) (puncta number/10 μm length dendrite close to the soma). Images (PDM) were captured for co-localization analysis using Image J software (NIH), and SynPAnal software [42] was used to analyze puncta density (L). A suitable threshold was set and maintained for images across the whole data set; the threshold was chosen to maximize the number of puncta while excluding noise, followed by selection of the appropriate color channel. Areas of interest (10 μm length of dendrite proximal to the soma) were selected and SynPAnal software was used to automatically compute puncta densities (L), based on the specified threshold settings. The software was able to distinguish whether individual pixels was part if a given puncta, and could distinguish between adjacent puncta.

In this study, the puncta densities of NMDARs on 10 μm length of dendrite close to the soma were evaluated. NMDARs that colocalized with synapsin I in dendrites were classified as synaptic NMDARs; all NMDARs found on dendrites were considered to be dendritic NMDARs.

Western blot

Primary hippocampal cells were briefly sonified in Tris HCl (50 mM, pH 7.4), containing 5 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulphate (SDS), protease inhibitors, and 150 mM NaCl, before clearing by centrifugation. Protein concentrations were determined using the DC protein assay (BioRad). After boiling, samples were electrophoresed (8% SDS polyacrylamide) and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight at 4°C with the following primary antibodies GluN1 (1:1000), GluN2A (1:1000), GluN2B (1:1000), SAP102 (1:500), GluN2A/Tyr¹³²⁵ (1:1000), GluN2B/pTyr¹⁴⁷² (1:1000), Fyn (1:1000), Src (1:1000), β-actin (1:4000), before incubation with HRP-conjugated anti-mouse or anti-rabbit antibodies (1:3000). Protein bands were visualized with ECL Plus (Amersham Biosciences), and signals were quantified using ImageJ software. Band density values were normalized to β-actin after subtracting background in each region of interest, and then to values from the corresponding controls.

Statistical analysis

Data are depicted as mean ± S.E.M. (5 indepen-dent experiments). Statistical analysis involved Independent-Sample t-test. A level of p < 0.05 was considered to be statistically significant.

RESULTS

Aβ acutely downregulates synaptic GluN2A

Aβ_1-40 is one of the two most abundant species of amyloid deposits in the brain of Alzheimer’s patients. Aβ_1-40 can stably assemble into soluble low molecular oligomers [43], and the soluble prefibrillar oligomers are recognized as the main species responsible for synaptic dysfunction [44, 45].

In the present experiment, Aβ_1-40 was diluted in culture medium and incubated at 37°C for 1 h. The oligomeric nature of Aβ_1-40 used to treat neurons was confirmed by AFM (Fig. 1). Spheroid or globular oligomers of similar size were detected. With section analysis, the average Z height of the probe was 3.1 nm, corresponding to the predicted size of soluble oligomers [46].

To quantify synaptic NMDAR puncta, double-labeling with antibodies against NMDAR and synapsin I were used. Synaptic and non-synaptic NMDARs puncta were distinguishable with the aid of Image J and SynPAnal softwares (Fig. 2). NMDARs were detected in the cytoplasm, dendritic shafts and spines. Those NMDAR clusters in dendrites that were not colocalized with the presynaptic marker synapsin I were considered to represent non-synaptic NMDARs.

Treatment of neurons with Aβ_1-40 (10 μM, 1 h) resulted in differential distributions of NMDARs at synaptic and non-synaptic sites; the treatment also altered total protein levels of the different NMDARs (Table 1).

When immunocytochemical methods were used to monitor GluN2A puncta density, our results show that Aβ_1-40 (10 μM, 1 h) treatment induced a significant reduction puncta densities of dendritic GluN2A (Table 1, Fig. 3A, D; Aβ, 160.3 ± 3.4 versus control, 209.6 ± 3.7 puncta/10 μm dendritic length, p < 0.01) and synaptic GluN2A in 10 μm dendritic length close to the soma (Table 1, Fig. 3C, D; Aβ, 30.5 ± 1.5 versus control, 71.7 ± 2.5 puncta/10 μm dendritic length, p < 0.01). SynapsinI (Fig. 3B, D; Aβ: 176.1 ± 16.4 versus control: 190.2 ± 16.4 puncta/10 μm dendritic length, p > 0.05) puncta density was not affected by exposure to Aβ. At the same time, the treatment does not alter total GluN2A protein levels in immunoblots of cell lysates (Table 1, Fig. 3E; Aβ: 110.5 ± 30.3% of control, p > 0.05).

Differential regulation of GluN2B and SAP102 by Aβ

The GluN2B subunit of the NMDAR is highly expressed at birth but its levels gradually diminish with age, as the expression of the GluN2A subunit increases [47 –49]. Like GluN2A, this subunit binds glutamate and it is widely considered that the ratio of GluN2A:GluN2B subunits shifts during development and aging [50, 51]. The expression of GluN2B and GluN2A show similar levels of decline in AD [52 –54]. During synaptogenesis, GluN2B trafficking is facilitated by SAP102 [55].

In our present study, Aβ induced a significant increase of puncta densities of dendritic GluN2B (Table 1, Fig. 4A, D; Aβ: 194.2 ± 9.7 versus control: 169.6 ± 7.7 puncta/10 μm dendritic length, p < 0.05) and synaptic GluN2B (Table 1, Fig. 4C, D; Aβ: 119.2 ± 7.9 versus control: 70.5 ± 5.6 puncta/10 μm dendritic length, p < 0.05). In addition, Aβ-induced decreases in GluN2B protein levels in cell lysate were found by immunoblotting (Table 1, Fig. 4F; 83.8 ± 4.7% of control, p < 0.05).

Aβ led to a significant reduction in SAP102 puncta density (Fig. 4E; Aβ, 123.3 ± 2.5 versus control, 163.2 ± 2.5 puncta/10 μm dendritic length, p < 0.01) when dendrities of Aβ-treated neurons were examined. At the same time, Aβ did not influence SAP102 protein expression levels (Fig. 4G; 99.9 ± 12.8% of control, p > 0.05), as measured by immunoblotting. In contrast to the increased puncta densities of dendritic or synaptic GluN2B, the presently observed reduction of SAP102 puncta density in dendrites suggests that SAP102 does not directly affect anchoring of NR2B at synaptic sites.

Aβ leads to differential phosphorylation of GluN2A (pTyr¹³²⁵) and GluN2B (pTyr¹⁴⁷²)

Tyrosine phosphorylation is thought to influence the trafficking of GluN2 subunits [56, 57]. Immunocytochemical analysis revealed that Aβ treatment leads to a significant downregulation of dendritic pTyr¹³²⁵-GluN2A puncta density (Table 1, Fig. 5A, D; Aβ: 170.8 ± 1.9 versus control: 185.5 ± 2.8/10 μm dendritic length, p < 0.05) and a significant increase in synaptic pTyr¹³²⁵-GluN2A puncta density (Table 1, Fig. 5C, D; Aβ: 136.2 ± 2.8 versus control: 109.8 ± 2.5/10 μm dendritic length, p < 0.01).

Comparing to pTyr¹³²⁵-GluN2A, Aβ downregulates puncta densities of both dendritic (Table 1, Fig. 6A, D; Aβ: 179.0 ± 3.1 versus control: 199.0 ± 3.4/10 μm dendritic length, p < 0.05) and synaptic (Table 1, Fig. 6C, D; Aβ: 126.2 ± 2.3 versus control: 141.5 ± 2.5, p < 0.01) pTyr¹⁴⁷²-GluN2B.

At the same time, immunoblotting revealed that exposure of hippocampal neurons to 10 μM Aβ_1-40 for 1 h leads to a significant decrease in the total protein levels of phosphorylated GluN2B (Tyr residue 1472; 84.6 ± 2.6% of control, p < 0.05) (Table 1, Fig. 7B), but not of phosphorylated GluN2A (Tyr residue 1325; 94.7 ± 3.0% of control, p > 0.05) (Table 1, Fig. 7A).

In light of previous studies that showed tyrosine phosphorylation of GluN2A and GluN2B to be mediated by Src and Fyn, respectively [58], we monitored the protein levels of these kinases by immunoblotting to corroborate the changes seen in Fig. 7A and B. Consistent with the data shown in Fig. 7A and B, Aβ treatment did not alter Src expression (Fig. 7C; 101.9 ± 0.4% of control, p > 0.05) and resulted in a significant decrease in Fyn levels (Fig. 7D; 89.2 ± 0.9% of control, p < 0.05).

Aβ treatment does not influence GluN1 subunit expression

The GluN1 is an obligatory component of NMDAR; each NMDAR receptor complex contains two GluN1 subunits which bind the glutamate co-agonist,gylcine.

Exposure of primary hippocampal neurons to 10 μM Aβ_1-40 oligomers for 1 h did not alter dendritic and synaptic GluN1 puncta densities (Table 1, Fig. 8A, D; dendritic GluN1, Aβ: 174.9 ± 3.1 versus control:187.3 ± 6.18 puncta/10 μm dendritic length, p > 0.05; Fig. 8C, D; synaptic GluN1, Aβ: 112.7 ± 3.1 versus control: 115.1 ± 8.3 puncta/10 μm dendritic length, p > 0.05) or GluN1 protein levels of expression (Table 1, Fig. 8E; 92.4 ± 18.6% of control, p > 0.05), as evaluated by the complementary methods of immunocytochemistry and immunoblotting.

DISCUSSION

The important role of Aβ oligomers in synaptic dysfunction and neuronal loss in AD is indisputable. In addition, there is strong evidence that the neurotoxic actions of Aβ depend on activated NMDAR [19, 59], whose expression is altered in AD patient brains [60 –64]. We and others have shown that synaptic dysfunction is an early event induced by Aβ oligomers [15, 33]. However, the exact contribution of specific NMDAR subunits or subpopulations to AD pathology, especially during early stages of the disease, is unclear. This is an especially important issue in light of an increasing recognition that the pathological processes underpinning AD may precede clinical symptoms by many years [65, 66]. The latter suggests the existence of an approximately 50-year window of opportunity during which preventive or delay strategies can be initiated, well before the appearance of clinically-detectable symptoms [66]. The present in vitro experiments used cultured hippocampal cells that were obtained from early postnatal rats (aged 4 days), which after DIV7 display mature networks and functional synapses. Mature neurons in culture are characterized by extensive dendritic arborization and expression of “mature” GluN1/GluN2A receptors puncta in the vicinity of dendrite spines, mostly colocalized with synaptic proteins [22 , 67–69]. Here, we found that NMDA receptors are mainly clustered in the soma and proximal dendritic shaft, and that GluN2A subunits are abundantly located at non-synaptic sites (i.e. not colocalized with the synaptic protein, synapsin I).

GluN2A and GluN2B show dynamic changes, and both are critical regulators of NMDAR localization and function, with significant roles in learning and memory [70, 71]. Excitatory synaptic transmission is tightly regulated by the number of synaptic NMDAR, and Aβ is known to modulate synaptic NMDAR activity [72 –74]. Our finding supports the view that Aβ induces a shunting of GluN2A and GluN2B between synaptic and non-synaptic membrane sites and/or the cytoplasmic compartment of neurons [35] from early stages of Aβ pathology. Accordingly, the altered localization of GluN2A and GluN2B at dendrites in close proximity to the soma may be a key event in the early stages of AD.

The present results show that puncta densities of dendritic and synaptic GluN2A are significantly reduced after Aβ treatment. Since GluN2A are normally stabilized at the synapse by binding to the scaffold protein PSD-95 [23], the observed reduction in GluN2A after Aβ may be explained by our previous finding that Aβ decreases PSD-95 puncta density [33]. The loss of synaptic GluN2A would result in lower spine concentrations of Ca^2 + and thus, decreased synaptic strength and efficacy [23]. Previous work has shown that tyrosine phosphorylation is an important mechanism for the potentiation of NMDAR activity [75]. The present results, showing decreased puncta density of dendritic pTyr1325-GluN2A is consistent with reduced dendritic GluN2A puncta density under control conditions. The finding that Aβ_1-40 treatment increases synaptic pTyr1325-GluN2A puncta density is therefore likely to represent an early response. On the other hand, the lack of change in total protein levels (western blotting) may indicate a redistribution of GluN2A and pTyr¹³²⁵-GluN2A away from synaptic sites and toward to non-synaptic site or other subcellular compartments (e.g., distal dendrites, cytoplasm, membrane of the cell body, axons). Further, a contribution of glial GluN2A/pTyr¹³²⁵-GluN2A [76] to the total levels of this protein measured cannot be excluded.

Besides, our results that Aβ_1-40 rapidly downregulates NR2B protein level is consistent with that of some reports on the effects of soluble Aβ oligomers in in vitro and in vivo experiments [77, 78]. The Aβ-induced loss of GluN2B is likely to affect synaptic plasticity [61, 79] and may explain why chronic treatment with the GluN2B antagonist piperidine18 does not protect against synapse loss in an Aβ-overexpressing mouse model of AD [80]. At the same time, we unexpectedly found increased puncta densities of dendritic and synaptic GluN2B; these may reflect a compensatory response to the reductions of total GluN2B levels following exposure to Aβ, adding support for the idea that dendrites that are close to the soma are particularly functionally sensitive to Aβ.

In addition to reducing GluN2B protein levels, Aβ treatment of hippocampal neurons also reduced the protein levels of pTyr¹⁴⁷²-GluN2B, as well as dendritic and synaptic puncta densities. Phosphorylation of the Tyr¹⁴⁷² epitope is reported to be associated with enrichment of synaptic GluN2B [81]. The present inconsistent changes of increased GluN2B and decreased pTyr¹⁴⁷²-GluN2B puncta densities, may suggest that phosphorylation of Tyr¹⁴⁷²-GluN2B is not required for translocation of GluN2B to the synapse [81], and the related regulation mechanism between GluN2B and phosphorylation of Tyr1472 may be more complex at synaptic sites. In addition, it should be noted that the postsynaptic GluN2B is a substrate for Fyn [82], and that phosphorylation of GluN2B at Tyr1472 strengthens the interaction between GluN2B and PSD-95 [83], a mechanism that links NMDAR signaling with Aβ toxicity. Further, tau protein, which plays an important role in the pathology of AD, was recently shown to be involved in the postsynaptic targeting of Fyn in dendrites [84, 85], while the accumulation of hyperphosphorylated tau within dendritic spines disrupts NMDAR anchoring and trafficking [86]. Whether the reductions of Fyn and pTyr¹⁴⁷²-GluN2B found here are related to alterations in tau hyperphosphorylation and relocation from axons to dendrites is a question for future investigations. Meanwhile, it would appear from the present results that reductions of GluN2B-pTyr¹⁴⁷² -GluN2B-Fyn protein levels precede the trafficking of hyperphosphorylated tau to dendritic spines.

This study also examined the potential role of SAP102, an important postsynaptic scaffolding protein, in mediating the actions of Aβ. SAP102 is highly expressed in adult human brain where it plays an important role in synaptic function, plasticity and regulation of NMDAR trafficking [2, 87]. Like glutamate receptors, postsynaptic scaffold proteins (e.g., PSD-95, SAP102) are also dynamically altered [88]. We here found that Aβ does not lead to significant alterations in the levels of SAP102 protein; however, Aβ did result in significantly smaller puncta density in dendrites. The non-tandem alterations in GluN2B and SAP102 likely suggests that SAP102 does not play a direct role in anchoring GluN2B at synaptic sites, although previous studies indicated preferential interactions between GluN2B and SAP102 [55, 89]. Notably, a recent study revealed a non-scaffolding role for SAP102 in clearing GluN2B from synapses [90]; thus, the decreased SAP102 puncta density in dendrites may lead to the reduced clearance of GluN2B from synapse sites. On the other hand, the finding that total SAP102 protein levels are unchanged could indicate that SAP102 relocalizes to the cytoplasm where it participates in GluN2B trafficking.

In conclusion, our findings demonstrate that Aβ exerts complex and distinct regulatory effects on the trafficking and phosphorylation of GluN2A and GluN2B, as well as on their localization within synaptic and non-synaptic sites. Increased understanding of these early events in Aβ-induced synaptic dysfunction is likely to be important for the development of timely preventive and therapeutic interventions.

Footnotes

ACKNOWLEDGMENTS

This work was supported by the Program for New Century Excellent Talents in University (NCET-10-0015), the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions (CIT&TCD201304175), Beijing Natural Science Foundation (5152004), National Natural Science Foundation of China (81301100, 81200968), and the Science and Technology Development Project of Beijing Education Committee (SQKM201210025003), as well as the EU-funded project SwitchBox (Contract 259772).

Authors’ disclosures available online ().

References

Terry

, Masliah

, Salmon

, Butters

, DeTeresa

, Hill

, Hansen

, Katzman

(1991) Physical basis of cognitive alterations in Alzheimer’s disease: Synapse loss is the major correlate of cognitive impairment. Ann Neurol 30, 572–580.

Proctor

, Coulson

, Dodd

(2010) Reduction in post-synaptic scaffolding PSD-95 and SAP-102 protein levels in the Alzheimer inferior temporal cortex is correlated with disease pathology. J Alzheimers Dis 21, 795–811.

Price

, Varghese

, Sowa

, Yuk

, Brautigam

, Ehrlich

, Dickstein

(2014) Altered synaptic structure in the hippocampus in a mouse model of Alzheimer’s disease with soluble amyloid-β oligomers and no plaque pathology. Mol Neurodegener 9, 41.

, Okamoto

, Lipton

, Xu

(2014) Oligomeric Aβ-induced synaptic dysfunction in Alzheimer’s disease. Mol Neurodegener 9, 48.

Mattson

, Cheng

, Davis

, Bryant

, Lieberburg

, Rydel

(1992) Beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci 12, 376–389.

Noda

, Nakanishi

, Akaike

(1999) Glutamate release from microglia via glutamate transporter is enhanced by amyloid-beta peptide. Neuroscience 92, 1465–1474.

Danysz

, Parsons

(2002) Neuroprotective potential of ionotropic glutamate receptor antagonists. Neurotox Res 4, 119–126.

Talantova

, Sanz-Blasco

, Zhang

, Xia

, Akhtar

, Okamoto

, Dziewczapolski

, Nakamura

, Cao

, Pratt

, Kang

, Tu

, Molokanova

, McKercher

, Hires

, Sason

, Stouffer

, Buczynski

, Solomon

, Michael

, Powers

, Kelly

, Roberts

, Tong

, Fang-Newmeyer

, Parker

, Holland

, Zhang

, Nakanishi

, Chen

, Wolosker

, Wang

, Parsons

, Ambasudhan

, Masliah

, Heinemann

, Piña-Crespo

, Lipton

(2013) Aβ induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss. Proc Natl Acad SciU S A 110, E2518–E2527.

Bitan

, Vollers

, Teplow

(2003) Elucidation of primary structure elements controlling early amyloid beta-protein oligomerization. J Biol Chem 278, 34882–34889.

10.

Anekonda

, Quinn

, Harris

, Frahler

, Wadsworth

, Woltjer

(2011) L-type voltage-gated calcium channel blockade with isradipine as a therapeutic strategy for Alzheimer’s disease. Neurobiol Dis 41, 62–70.

11.

Di Carlo

, Minicozzi

, Foderà

, Militello

, Vetri

, Morante

, Leone

(2015) Thioflavin T templates amyloid β(1-40) conformation and aggregation pathway. Biophys Chem 206, 1–11.

12.

Zhang

, Li

, Liu

, Song

, Li

, Yin

, Xiao

, Li

, Jiang

, Zong

, Zhang

, Wang

(2015) Protective effects of low molecular weight chondroitin sulfate on amyloid beta (Aβ)-induced damage in vitro and in vivo . Neuroscience 305, 169–182.

13.

Lue

, Kuo

, Roher

, Brachova

, Shen

, Sue

, Beach

, Kurth

, Rydel

, Rogers

(1999) Soluble amyloid-peptide concentration as a predictor of synaptic change in Alzheimer’s disease. Am J Pathol 155, 853–862.

14.

McLean

, Cherny

, Fraser

, Fuller

, Smith

, Beyreuther

, Bush

, Masters

(1999) Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer’s disease. Ann Neurol 46, 860–866.

15.

Roselli

, Tirard

, Lu

, Hutzler

, Lamberti

, Livrea

, Morabito

, Almeida

OFX

(2005) Soluble beta amyloid1-40 induces NMDA dependent degradation of postsynaptic density95 at glutamatergic synapses. J Neurosci 25, 11061–11070.

16.

Lacor

, Buniel

, Furlow

, Clemente

, Velasco

, Wood

, Viola

, Klein

(2007) Aβ oligomer induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer’s diseases. J Neurosci 27, 796–807.

17.

Barria

, Malinow

(2005) NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII. Neuron 48, 289–301.

18.

Bai

, Aida

, Yanagisawa

, Katou

, Sakimura

, Mishina

, Tanaka

(2013) NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina. Mol Brain 6, 34.

19.

Shimazawa

, Inokuchi

, Okuno

, Nakajima

, Sakaguchi

, Kato

, Oku

, Sugiyama

, Kudo

, Ikeda

, Takeda

, Hara

(2008) Reduced retinal function in amyloid precursor protein overexpressing transgenic mice via attenuating glutamate N-methyl-daspartate receptor signaling. J Neurochem 107, 279–290.

20.

Bellone

, Nicoll

(2007) Rapid bidirectional switching of synaptic NMDA receptors. Neuron 55, 779–785.

21.

Barria

, Malinow

(2002) Subunit-specific NMDAreceptor trafficking to synapses. Neuron 35, 345–354.

22.

Tovar

and Westbrook

(1999) The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro . J Neurosci 19, 4180–4188.

23.

Carroll

, Zukin

(2002) NMDA-receptor trafficking and targeting: Implications for synaptic transmission and plasticity. Trends Neurosci 25, 571–577.

24.

Tovar

, Westbrook

(2002) Mobile NMDA receptors at hippocampal synapses. Neuron 34, 255–264.

25.

Groc

, Choquet

(2006) AMPA and NMDA glutamate receptor trafficking: Multiple roads for reaching and leaving the synapse. Cell Tissue Res 326, 423–438.

26.

Snyder

, Nong

, Almeida

, Paul

, Moran

, Choi

, Nairn

, Salter

, Lombroso

, Gouras

, Greengard

(2005) Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8, 1051–1058.

27.

Vissel

, Krupp

, Heinemann

, Westbrook

(2001) A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux. Nat Neurosci 4, 587–596.

28.

Wang

, Salter

(1994) Regulation of NMDA receptors by tyrosine kinases and phosphatases. Nature 369, 233–235.

29.

Lau

, Huganir

(1995) Differential tyrosine phosphorylation of N-methyl-D-aspartate receptor subunits. J Biol Chem 270, 20036–20041.

30.

Nakazawa

, Komai

, Tezuka

, Hisatsune

, Umemori

, Semba

, Mishina

, Manabe

, Yamamoto

(2001) Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor. J Biol Chem 276, 693–699.

31.

Taniguchi

, Nakazawa

, Tanimura

, Kiyama

, Tezuka

, Watabe

, Katayama

, Yokoyama

, Inoue

, Izumi-Nakaseko

, Kakuta

, Sudo

, Iwakura

, Umemori

, Inoue

, Murphy

, Hashimoto

, Kano

, Manabe

, Yamamoto

(2009) Involvement of NMDAR2A tyrosine phosphorylation in depression-related behaviour. EMBO J 28, 3717–3729.

32.

Yang

, Leonard

(2001) Identification of mouse NMDA receptor subunit NR2A C-terminal tyrosine sites phosphorylated by coexpression with v-Src. J Neurochem 77, 580–588.

33.

Liu

, Chang

, Roselli

, Almeida

, Gao

, Wang

, Yew

, Wu

(2010) Amyloid-β induces caspase-dependent loss of PSD-95 and synaptophysin through NMDA receptors. J Alzheimers Dis 22, 541–556.

34.

Crochemore

, Lu

, Wu

, Liposits

, Sousa

, Holsboer

, Almeida

OFX

(2005) Direct targeting of hippocampal neurons for apoptosis by glucocorticoida is reversible by mineralocorticoid receptor activation. Mol Psychiatry 10, 790–798.

35.

Roselli

, Hutzler

, Wegerich

, Livrea

, Almeida

OFX

(2009) Disassembly of shank and homer synaptic clusters is driven by soluble beta-amyloid (1-40) through divergent NMDAR-dependent signalling pathways. PLoS One 4, e6011.

36.

Roselli

, Livrea

, Almeida

OFX

(2011) CDK5 is essential for soluble amyloid β-induced degradation of GKAP and remodeling of the synaptic actin cytoskeleton. PLoS One 6, e23097.

37.

Walsh

, Lomakin

, Benedek

, Condron

, Teplow

(1997) Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. J Biol Chem 272, 22364–22372.

38.

Bitan

, Lomakin

, Teplow

(2001) Amyloid protein oligomerization prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins. J Biol Chem 276, 35176–35184.

39.

Stine

, Dahlgren

, Krafft

, LaDu

(2003) In vitro characterization of conditions for amyloid- peptide oligomerization and fibrillogenesis. J Biol Chem 278, 11612–11622.

40.

O’Nuallain

, Williams

, Westermark

, Wetzel

(2004) Seeding specificity in amyloid growth induced by heterologous fibrils. J Biol Chem 279, 17490–17499.

41.

Wogulis

, Wright

, Cunningham

, Chilcote

, Powell

, Rydel

(2005) Nucleation-dependent polymerization is an essential component of amyloid-mediated neuronal cell death. J Neurosci 25, 1071–1080.

42.

Danielson

, Lee

(2014) SynPAnal: Software for rapid quantification of the density and intensity of protein puncta from fluorescence microscopy images of neurons. PLoS One 9, e115298.

43.

Bitan

, Kirkitadze

, Lomakin

, Vollers

, Benedek

, Teplow

(2003) Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways. Proc Natl Acad Sci U S A 100, 330–335.

44.

Medeiros

, Prediger

, Passos

, Pandolfo

, Duarte

, Franco

, Dafre

, Di Giunta

, Figueiredo

, Takahashi

, Campos

, Calixto

(2007) Connecting TNF-alpha signaling pathways to iNOS expression in a mouse model of Alzheimer’s disease: Relevance for the behavioral and synaptic deficits induced by amyloid beta protein. J Neurosci 27, 5394–5404.

45.

Prediger

, Franco

, Pandolfo

, Medeiros

, Duarte

, Di Giunta

, Figueiredo

, Farina

, Calixto

, Takahashi

, Dafre

(2007) Differential susceptibility following beta-amyloid peptide-(1–40) administration in C57BL/6 and Swiss albino mice: Evidence for a dissociation between cognitive deficits and the glutathione system response. Behav Brain Res 177, 205–213.

46.

Chromy

, Nowak

, Lambert

, Viola

, Chang

, Velasco

, Jones

, Fernandez

, Lacor

, Horowitz

, Finch

, Krafft

, Klein

(2003) Self-assembly of Abeta(1-42) into globular neurotoxins. Biochemistry 42, 12749–12760.

47.

Monyer

, Burnashev

, Laurie

, Sakmann

, Seeburg

(1994) Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529–540.

48.

Sheng

, Cummings

, Roldan

, Jan

(1994) Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368, 144–147.

49.

Chang

, Liu

, Zhang

, Wang

, Gao

, Wu

(2009) Different expression of NR2B and PSD-95 in rat hippocampal subregions during postnatal development. Microsc Res Tech 72, 517–524.

50.

Philpot

, Sekhar

, Shouval

, Bear

(2001) Visual experience and deprivation bidirectionally modify the composition and function of NMDA receptors in visual cortex. Neuron 29, 157–169.

51.

Sanz-Clemente

, Nicoll

, Roche

(2013) Diversity in NMDA receptor composition, many regulators, many consequences. Neuroscientist 19, 62–75.

52.

Magnusson

(2000) Declines in mRNA expression of different subunits may account for differential effects of aging on agonist and antagonist binding to the NMDA receptor. J Neurosci 20, 1666–1674.

53.

Magnusson

, Nelson

, Young

(2002) Age-related changes in the protein expression of subunits of the NMDA receptor. Brain Res Mol Brain Res 99, 40–45.

54.

Zamzow

, Elias

, Shumaker

, Larson

, Magnusson

(2013) An increase in the association of GluN2B containing NMDA receptors with membrane scaffolding proteins was related to memory declines during aging. J Neurosci 33, 12300–12305.

55.

Elias

, Elias

, Apostolides

, Kriegstein

, Nicoll

(2008) Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development. Proc Natl Acad Sci U S A 105, 20953–20958.

56.

Chung

, Huang

, Lau

, Huganir

(2004) Regulation of the NMDA receptor complex and trafficking by activity-dependent phosphorylation of the NR2B subunit PDZ ligand. J Neurosci 24, 10248–10259.

57.

Chen

, Roche

(2007) Regulation of NMDA receptors by phosphorylation. Neuropharmacology 53, 362–368.

58.

Salter

, Kalia

(2004) Src kinases, a hub for NMDA receptor regulation. Nat Rev Neurosci 5, 317–328.

59.

Danysz

, Parsons

, Mobius

, Stoffler

, Quack

(2000) Neuroprotective and symptomatological action of memantine relevant for Alzheimer’s disease–a unified glutamatergic hypothesis on the mechanism of action. Neurotox Res 2, 85–97.

60.

Sze

, Bi

, Kleinschmidt-DeMasters

, Filley

, Martin

(2001) N-Methyl-D-aspartate receptor subunit proteins and their phosphorylation status are altered selectively in Alzheimer’s disease. J Neurol Sci 182, 151–159.

61.

Ulas

, Cotman

(1997) Decreased expression ofN-methyl-D-aspartate receptor 1 messenger RNA in select regions of Alzheimer brain. Neuroscience 79, 973–982.

62.

Hynd

, Scott

, Dodd

(2004) Differential expression of N-methyl-D-aspartate receptor NR2 isoforms in Alzheimer’s disease. J Neurochem 90, 913–919.

63.

Mishizen-Eberz

, Rissman

, Carter

, Ikonomovic

, Wolfe

, Armstrong

(2004) Biochemical and molecular studies of NMDA receptor subunits NR1/2A/2B in hippocampal subregions throughout progression of Alzheimer’s disease pathology. Neurobiol Dis 15, 80–92.

64.

Mohamed

, Lee

, Francis

, Esiri

, Chen

, Lai

(2015) Differential alterations of neocortical GluN receptor subunits in patients with mixed subcortical ischemic vascular dementia and Alzheimer’s disease. J Alzheimers Dis 44, 431–437.

65.

Braak

, Del Tredici

(2011) The pathological process underlying Alzheimer’s disease in individuals under thirty. Acta Neuropathol 121, 171–181.

66.

Calderón-Garcidueñas

, Franco-Lira

, Mora-Tiscareño

, Medina-Cortina

, Torres-Jardón

, Kavanaugh

(2013) Early Alzheimer’s and Parkinson’s disease pathology in urban children: Friend versus Foe responses-it is time to face the evidence. Biomed Res Int 2013, 161687.

67.

Rao

, Kim

, Sheng

, Craig

(1998) Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture. J Neurosci 18, 1217–1229.

68.

Liao

, Zhang

, O’Brien

, Ehlers

, Huganir

(1999) Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons. Nat Neurosci 2, 37–43.

69.

Pérez-Otaño

, Ehlers

(2004) Learning from NMDA receptor trafficking, clues to the development and maturation of glutamatergic synapses. Neurosignals 13, 175–189.

70.

Tang

, Shimizu

, Dube

, Rampon

, Kerchner

, Zhuo

, Liu

, Tsien

(1999) Genetic enhancement of learning and memory in mice. Nature 401, 63–69.

71.

Tang

, Schuman

(2002) Protein synthesis in the dendrite. Philos Trans R Soc Lond B Biol Sci 357, 521–529.

72.

Palop

, Mucke

(2010) Amyloid-beta-induced neuronal dysfunction in Alzheimer’s disease, from synapses toward neural networks. Nat Neurosci 13, 812–818.

73.

Texidó

, Martín-Satué

, Alberdi

, Solsona

, Matute

(2011) Amyloid β peptide oligomers directly activate NMDA receptors. Cell Calcium 49, 184–190.

74.

Molokanova

, Akhtar

, Sanz-Blasco

, Tu

, Piña-Crespo

, McKercher

, Lipton

(2014) Differential effects of synaptic and extrasynaptic NMDA receptors on Aβ-induced nitric oxide production in cerebrocortical neurons. J Neurosci 34, 5023–5028.

75.

Goebel

, Alvestad

, Coultrap

, Browning

(2005) Tyrosine phosphorylation of the N-methyl-D-aspartate receptor is enhanced in synaptic membrane fractions of the adult rat hippocampus. Brain Res Mol Brain Res 142, 65–79.

76.

Zhou

, Li

, Zhao

, Yang

, Dong

, Yue

, Ma

, Wang

, Chen

, Cui

, Yu

(2010) Astrocytes express N-methyl-D-aspartate receptor subunits in development, ischemia and post-ischemia. Neurochem Res 35, 2124–2134.

77.

Rönicke

, Mikhaylova

, Rönicke

, Meinhardt

, Schröder

, Fändrich

, Reiser

, Kreutz

, Reymann

(2011) Early neuronal dysfunction by amyloid β oligomers depends on activation of NR2B-containing NMDA receptors. Neurobiol Aging 32, 2219–2228.

78.

Brouillette

, Caillierez

, Zommer

, Alves-Pires

, Benilova

, Blum

, De Strooper

, Buée

(2012) Neurotoxicity and memory deficits induced by soluble low-molecular-weight amyloid-β1-42 oligomers are revealed in vivo by using a novel animal model. J Neurosci 32, 7852–7861.

79.

Proctor

, Coulson

, Dodd

(2011) Post-synaptic scaffolding protein interactions with glutamate receptors in synaptic dysfunction and Alzheimer’s disease. Prog Neurobiol 93, 509–521.

80.

Hanson

, Meilandt

, Gogineni

, Reynen

, Herrington

, Weimer

, Scearce-Levie

, Zhou

(2014) Chronic GluN2B antagonism disrupts behavior in wild-type mice without protecting against synapse loss or memory impairment in Alzheimer’s disease mouse models. J Neurosci 34, 8277–8288.

81.

Goebel-Goody

, Davies

, Alvestad Linger

, Freund

, Browning

(2009) Phospho-regulation of synaptic and extrasynaptic N-methyl-d-aspartate receptors in adult hippocampal slices. Neuroscience 158, 1446–1459.

82.

Nakazawa

, Komai

, Tezuka

, Hisatsune

, Umemori

, Semba

, Mishina

, Manabe

, Yamamoto

(2001) Characterization of Fyn mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor. J Biol Chem 276, 693–699.

83.

Rong

, Lu

, Bernard

, Khrestchatisky

, Baudry

(2001) Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95. J Neurochem 79, 382–390.

84.

Haass

, Mandelkow

(2010) Fyn-tau-amyloid: A toxic triad. Cell 142, 356–358.

85.

Ittner

, Ke

, Delerue

, Bi

, Gladbach

, van Eersel

, Wölfing

, Chieng

, Christie

, Napier

, Eckert

, Staufenbiel

, Hardeman

, Götz

(2010) Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer’s disease mouse models. Cell 142, 387–397.

86.

Hoover

, Reed

, Su

, Penrod

, Kotilinek

, Grant

, Pitstick

, Carlson

, Lanier

, Yuan

, Ashe

, Liao

(2010) Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. Neuron 68, 1067–1081.

87.

Wei

, Behrman

, Wu

, Chen

(2015) Subunit-specific regulation of N-methyl-D-aspartate (NMDA) receptor trafficking by SAP102 protein splice variants. J Biol Chem 290, 5105–5116.

88.

Newpher

, Ehlers

(2008) Glutamate receptor dynamics in dendritic microdomains. Neuron 58, 72–97.

89.

Prybylowski

, Chang

, Sans

, Kan

, Vicini

, Wenthold

(2005) The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2. Neuron 47, 845–857.

90.

Chen

, Gray

, Sanz-Clemente

, Wei

, Thomas

, Nicoll

, Roche

(2012) SAP102 mediates synaptic clearance of NMDA receptors. Cell Rep 2, 1120–1128.

Differential Regulation of N-Methyl-D-Aspartate Receptor Subunits is An Early Event in the Actions of Soluble Amyloid-β 1-40 Oligomers on Hippocampal Neurons

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Chemicals and reagents

Primary hippocampal neurons culture

Soluble Aβ1-40 oligomers preparation and treatment

Immunofluorescence

Puncta analysis

Western blot

Statistical analysis

RESULTS

Aβ acutely downregulates synaptic GluN2A

Differential regulation of GluN2B and SAP102 by Aβ

Aβ leads to differential phosphorylation of GluN2A (pTyr1325) and GluN2B (pTyr1472)

Aβ treatment does not influence GluN1 subunit expression

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Soluble Aβ_1-40 oligomers preparation and treatment

Aβ leads to differential phosphorylation of GluN2A (pTyr¹³²⁵) and GluN2B (pTyr¹⁴⁷²)