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Cell Calcium 51 (2012) 95– 106

Contents lists available at SciVerse ScienceDirect

Cell Calcium

j ourna l ho me page: www.elsev ier .com/ locate /ceca

myloid beta peptide 1–42 disturbs intracellular calcium homeostasis throughctivation of GluN2B-containing N-methyl-d-aspartate receptors in corticalultures

.L. Ferreiraa, L.M. Bajoucoa, S.I. Motaa, Y.P. Aubersonc, C.R. Oliveiraa,b, A.C. Regoa,b,∗

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, PortugalFaculty of Medicine, University of Coimbra, Coimbra, PortugalNovartis Institutes of Biomedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland

r t i c l e i n f o

rticle history:eceived 18 April 2011eceived in revised form 4 November 2011ccepted 17 November 2011vailable online 15 December 2011

eywords:lzheimer’s diseasemyloid beta peptide (A�)alcium-Methyl-d-aspartate receptorluN2A subunitluN2B subunit

a b s t r a c t

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that leads to debilitating cognitivedeficits. Recent evidence demonstrates that glutamate receptors are dysregulated by amyloid beta pep-tide (A�) oligomers, resulting in disruption of glutamatergic synaptic transmission which parallels earlycognitive deficits. Although it is well accepted that neuronal death in AD is related to disturbed intracel-lular Ca2+ (Ca2+

i) homeostasis, little is known about the contribution of NMDARs containing GluN2A orGluN2B subunits on A�-induced Ca2+

i rise and neuronal dysfunction. Thus, the main goal of this workwas to evaluate the role of NMDAR subunits in dysregulation of Ca2+

i homeostasis induced by A� 1–42preparation containing both oligomers (in higher percentage) and monomers in rat cerebral corticalneurons. The involvement of NMDARs was evaluated by pharmacological inhibition with MK-801 orthe selective GluN2A and GLUN2B subunit antagonists NVP-AAM077 and ifenprodil, respectively. Weshow that A�, like NMDA, increase Ca2+

i levels mainly through activation of NMDARs containing GluN2Bsubunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca2+

i rise induced by a high concentra-tion of A� (1 �M), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca2+

i

homeostasis. Moreover, A� modulate NMDA-induced responses and vice versa. Indeed, pre-exposure toA� (1 �M) decrease NMDA-evoked Ca2+

I rise and pre-exposure to NMDA decrease A� response. Inter-estingly, simultaneous addition of A� and NMDA potentiate Ca2+

I levels, this effect being regulated byGluN2A and GluN2B subunits in opposite manners. This study contributes to the understanding of themolecular basis of early AD pathogenesis, by exploring the role of GluN2A and GluN2B subunits in themechanism of A� toxicity in AD.

. Introduction

Alzheimer’s disease (AD) is the leading cause of dementian western countries and the most prevalent neurodegenerativeisease in the elderly population, affecting 26.6 million peopleorldwide [1]. Age-related forms of dementia lead to sporadic AD.onversely, less than 10% of cases are associated with familial AD,ue to mutations in either amyloid precursor protein, presenilin-1

r presenilin-2 genes. AD hallmarks include atrophy in the cortex,ippocampus and amygdala [2]. Neuropathologically, AD is char-cterized by senile plaques, composed of extracellular deposits of

∗ Corresponding author at: CNC-Center for Neuroscience and Cell Biology Univer-ity of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal.el.: +351 239 820190; fax: +351 239 822776.

E-mail addresses: a.cristina.rego@gmail.com, arego@fmed.uc.pt,crego@cnc.uc.pt (A.C. Rego).

143-4160/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2011.11.008

© 2011 Elsevier Ltd. All rights reserved.

amyloid-beta peptides (A�) and intracellular neurofibrillary tan-gles formed by hyperphosphorylated tau [2,3]. A� is produced byproteolytic cleavage of APP by sequential activity of �- and �-secretases, producing A�1–42 and A�1–40 [4,5].

Neurodegeneration and synaptic dysfunction induced by A�involves overactivation of the N-methyl-d-aspartate (NMDA)receptors (NMDARs) resulting in the elevation of intracellularCa2+

i levels, a process named excitotoxicity [6–10]. Activation ofNMDARs was hypothesized to occur at late-stage AD, when plaqueformation is expected. However, recent reports strongly suggestthat glutamate receptors are dysregulated by A� accumulationin the initial stages of AD, resulting in disruption of glutamater-gic synaptic transmission, which parallels early cognitive deficits[11]. Thus, early phases of AD (characterized by the presence

of A� monomers and oligomers) are linked to NMDAR-inducedsynaptic dysfunction, which appears to precede neurodegeneration[7,12–14]. Accordingly, recent studies suggest that A� oligomersare the main neurotoxic species involved early in AD [15,16].

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ndeed, oligomeric species were shown to be more toxic to corticaleurons than fibrillar forms, the main components of senile plaques17]. In addition, it has been reported that A�1–42 oligomers are

uch more prone to aggregation and are more neurotoxic thanhose composed by the A�1–40 peptide [5,18].

NMDAR functional downregulation is thought to take place dur-ng the initial stages of AD. Supporting this view, many studies haveuggested that A� may reduce surface GluN1 subunit of NMDARs,mpairing its function [19–21], leading to the depressed synapticlutamatergic transmission observed in AD.

Three families of genes (GluN1, GluN2 and GluN3) have beendentified that encode NMDAR subunits [22]. Functional NMDARsre heterotetramers composed of two glycine-binding GluN1ubunits and two glutamate-binding GluN2 (GluN2A-GluN2D)ubunits, or in some cases GluN3 (GluN3A and/or GluN3B) sub-nits, the latter replacing the GluN2 subunits [23]. The mostidely expressed NMDARs contain the obligatory subunit GluN1lus either GluN2B or GluN2A or a mixture of the two. GluN2Bnd GluN2D are expressed at high levels in early developmen-al stages (prenatally), whereas GluN2A and GluN2C expressions first detected near birth [24]. In adults, GluN2A is ubiqui-ously expressed in the brain, GluN2B is mostly restricted to theorebrain, GluN2C is restricted to the cerebellum, and GluN2Ds expressed in small numbers of cells in selected brain regions25].

It has been recently proposed that GluN2A- and GluN2B-ontaining NMDAR are linked to different intracellular cascades,articipating in different functions, from synaptic plasticity toathological conditions [26–28]. Although it is well accepted thateuronal death in AD is related to disturbed Ca2+

i homeostasisnvolving the NMDAR [6,10,29,30] and that early NMDAR dys-egulation occur in AD, little is known about the contribution ofhe GluN2A or GluN2B subunits on A�-induced neuronal dysfunc-ion. In this work we evaluated the contribution of the NMDARsubunits GluN2A and GluN2B, on Ca2+

i dysregulation induced byirect exposure to A�1–42. This study supports the hypothesishat GluN2A and/or GluN2B subunits of the NMDARs are impor-ant in the initial stages of AD pathogenesis and are involved ineuronal dysfunction induced by A�, leading to cortical neurode-eneration.

. Materials and methods

.1. Materials

Neurobasal medium and B27 supplement were purchasedrom GIBCO (Paisley, UK). Ifenprodi, resazurin and anti-�-tubulinas from Sigma Chemical Co. (St. Louis, MO, USA). NMDA was

btained from Tocris (Cookson, UK) and synthetic amyloid-beta–42 peptide from Bachem (Bubendorf, Switzerland). (+)-5-ethyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-iminealeate (MK-801) was obtained from Calbiochem (Darmstadt,ermany); [(R)-[(S)-1-(4-bromophenyl)-ethylamino]-(2,3-dioxo-,2,3,4 tetrahydroquinoxalin-5-yl)-me thyl]-phosphonic acidNVP-AAM077) was a generous gift from Novartis Pharma AG,asel, Switzerland. Sulfo-NHS-SS-biotin and NeutrAvidinTM werebtained from Pierce (Rockford, IL, USA). The antibody against theenatured form of GluN1 subunit was obtained from Cell Signaling,hereas anti-GluN2A and GluN2B were from Millipore Chemicon

Peniecula, CA, USA). Secondary antibodies conjugated to alkalinehosphatase (anti-mouse and anti-rabbit) were purchased from

mersham Biosciences (Buckinghamshire, UK). The fluorescencerobe Fura-2/AM and the antibody against transferrin receptorere obtained from Molecular Probes (Invitrogen, USA). All other

eagents were of analytical grade.

m 51 (2012) 95– 106

2.2. Primary cortical cultures

Primary cultures of rat cerebral cortex were prepared asdescribed previously [31] with some minor modifications. Briefly,frontal cerebral cortices free of meninges, were dissected out fromWistar fetal rats at embryonic 16 day and collected in Ca2+, Mg2+-free Krebs medium (120 mM NaCl, 4.33 mM KCl, 1.2 mM KH2PO4,25.5 mM NaHCO3, 13 mM glucose, 10 mM Hepes, pH 7.4), contain-ing 0.3% fatty acid-free BSA. Tissues were then treated with 0.035%trypsin plus 0.004% deoxyribonuclease I in BSA-Krebs medium, for7 min at 37 ◦C, followed by addition of 0.038% trypsin inhibitorin order to block enzymatic digestion. Cells were resuspended inKrebs medium and centrifuged at 1000 rpm for 5 min and thenplated at a density of 0.63 × 106 cells/cm2 in both poly-d-lysinecoated 96- or 6-well plates for intracellular Ca2+ measurementsand Western Blotting, respectively, in Neurobasal Medium supple-mented with 2% B27 supplement, 0.5 mM glutamine and 50 �g/mlgentamicin. Cells were cultured for 8 days, in a humidified incu-bator chamber with 95% air and 5% CO2 at 37 ◦C. Cortical culturesat 8 days in vitro (DIV) contained few glial cells (less than 10%) asassessed using antibodies against the neuronal marker microtubuleassociated protein-2 (MAP-2) and the marker of astrocytic prolifer-ation glial fibrillary acidic protein (GFAP) [32]. In cells cultured for15 days, half medium was changed at day 8 in culture. All animalexperiments were carried out following the Guide for laboratoryanimal practice of the Center for Neuroscience and Cell Biology,University of Coimbra, with care to minimize the number of animalsand their suffering.

2.3. Preparation of amyloid- peptide

A peptide preparation containing a high percentage of A�oligomers and monomers, previously described as ADDLs (Abeta-derived diffusible ligands), was made from synthetic A�1–42peptide (henceforward referred to as A�), as previously described[5,17]. Briefly, synthetic A� peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to a final concentration of 1 mM. HFIPwas then removed in a Speed Vac (Ilshin Lab. Co. Ltd., Ede, TheNetherlands), and dried HFIP film was stored at −20 ◦C. The pep-tide film was resuspended to make a 5 mM solution in anhydrousdimethyl sulfoxide. A� peptides were further prepared by dilutingthe solution in phenol red-free Ham’s F-12 medium without glu-tamine to a final concentration of 100 �M and incubated overnightat 4 ◦C. The preparation was centrifuged at 15,000 × g for 10 minat 4 ◦C to remove insoluble aggregates, and the supernatant con-taining soluble oligomers and monomers was transferred to cleantubes and stored at 4 ◦C. Protein concentrations of A� were thendetermined using the Bio-Rad protein dye assay reagent. Sam-ples containing 10 �g of protein were diluted (1:2) with samplebuffer (containing: 40% glycerol, 2% SDS, 0.2 M Tris–HCl, pH 6.8 and0.005% Coomassie G-250). The presence of different assembly pep-tide forms (monomers, oligomers and/or fibrils) in the preparationwas evaluated by 4–16% Tris–Tricine SDS-PAGE gel electrophoresisand further stained with Coomassie blue.

2.4. Western blot for GluN1, GluN2A and GluN2B subunits

Cellular extracts were performed in the embryo frontal cortex atday 0 (directly from the cortical tissue) or in cells cultured for 8 or15 DIV, using the Ripa buffer (containing 150 mM NaCl, 50 mM Tris,5 mM EGTA, 1% Triton X-100, 0.5% DOC, 0.1% SDS), supplementedwith 1 mM DTT, 1 mM PMSF and 1:1000 protease inhibitor cocktail

(chymostatin, pepstatin A, leupeptin and antipain). Protein contentwas determined by BioRad method, and the samples were denat-urated with 6 times concentrated denaturating buffer at 95 ◦C,for 5 min. Equivalent amounts of protein (15 �g) were separated

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n a 7% SDS-PAGE gel electrophoresis and electroblotted ontoolyvinylidene difluoride (PVDF) membranes. The membranesere further blocked with 5% fat-free milk and incubated with

ntibodies directed against GluN1 (1:1000), GluN2A (1:1000)r GluN2B (1:500) subunits. To control for loading of the gels,n anti-tubulin antibody was used. Immunoreactive bands wereisualized by alkaline phosphatase activity after incubation withCF reagent on a BioRad Versa Doc 3000 Imaging System.

.5. Cell surface biotinylation

Cortical cells cultured for 8 DIV were subjected to surfaceiotinylation in order to evaluate cell surface receptors, accordingo Kurup and collaborators [33] with some minor modifications.ells were rinsed twice in ice-cold PBS and further incubated in PBSontaining 1.5 mg/ml EZ-LinkTM sulfo-NHS-SS-biotin, for 20 min,t 4 ◦C. The non-bound biotin was removed by washing the cellsn PBS, and cell lysates were prepared in PBS containing proteasenhibitors, 0.1% SDS and 1% Triton X-100. The lysates were son-cated for 30 s and centrifuged for 5 min at 20,800 × g (4 ◦C); theupernatant was further incubated with 50 �l of NeutrAvidinTMlus beads, for 2 h, in a rotary shaker with gentle agitation, at◦C. Biotinylated proteins were washed 3 times at 3000 × g for

min (at 4 ◦C) and then eluted with denaturating buffer at 95 ◦C for min and centrifuged again at 20,800 × g, for 5 min, by using spin-

centrifuge tube (0.45 �m filter). Samples were then processed byestern Blotting for analysis of surface expression of GluN2A and

luN2B, as described above, and the transferrin receptor was useds a loading control.

.6. Alamar blue reducing assay

Cortical neurons were exposed to 0.5 or 1 �M soluble A�–42 preparation described above for 6 h, in conditioned culture

ig. 1. Expression of NMDA receptor subunits in cerebral cortical cultures. (A) Time-dep180 kDa) subunits of the NMDA receptors in cultured cortical cells isolated from 16 day-o

estern Blotting analysis of cell surface expression of GluN2A and GluN2B subunits labells cultured for 8 DIV. The transferrin receptor (Transferrin R, 90 kDa) was used as loanalysis: **p < 0.01, ***p < 0.01 significantly different when compared with subunit express

m 51 (2012) 95– 106 97

medium. When indicated, neurons were exposed to 100 �M NMDAplus 20 �M glycine in Mg2+-free Na+ medium (to selectively acti-vate the NMDARs), containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2,10 Glucose, 10 Hepes, pH 7.4/NaOH, for 15 min. In some exper-iments, after acute exposure to treatments, cells were incubatedfor further 18 h (‘recovery period’) in previously collected con-ditioned culture medium without the injury stimuli, in order toevaluate the delayed effects of A� or NMDA treatments. Phar-macological inhibition was achieved by incubating the cells inthe absence or presence of NMDA receptor antagonists, namelyifenprodil (10 �M), which selectively inhibits GluN2B-NMDARs,NVP-AAM077 (50 nM), a selective antagonist of GluN2A-NMDARsor MK-801 (10 �M), an antagonist of all NMDAR subtypes. In theconcentration used in this study, NVP-AAM077 is predicted toimpair activation of GluN2A-NMDARs, but not GluN2B-NMDARs[34,35], whereas ifenprodil selectively inhibits GluN2B-NMDARs[36]. In these cases, 5 min pre-incubation with NMDARs antago-nists was performed and maintained during treatment with NMDA.Alamar blue, a cell viability indicator that uses the natural reduc-ing ‘power’ of living cells to convert resazurin to the fluorescentmolecule resorufin, was added to treated or untreated cortical cellsin Na+ medium at a final concentration (1:100), for 1 h at 37 ◦C. Theabsorbance was detected at 570 nm (reference: 600 nm) by using amicroplate reader Spectra Max Plus 384 (Molecular Devices, USA)and results were normalized to the percentage of control (untreatedcells).

2.7. Intracellular Ca2+ recording

Cortical cultures were incubated in fresh Neurobasal medium

without added B27 supplement in the presence of the fluorescentprobe Fura-2/AM (10 �M) in the incubator chamber, at 37 ◦C for1 h. After a washing step, Ca2+

i levels were measured in cell pop-ulations subjected to NMDA (10 or 100 �M) or A� (0.5 or 1 �M)

endent total expression levels of GluN1 (120 kDa), GluN2A (180 kDa) and GluN2Bld rat embryos (0 days in vitro, DIV) or cultured for 8 or 15 days. (B) Representative

eled by biotinylation, followed by precipitation with neutravidin beads in corticalding control. Data are the mean ± S.E.M. of 3 independent experiments. Statisticalion at DIV 0 (Tukey’s post hoc test).

9 Calcium 51 (2012) 95– 106

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Fig. 2. Effect of NMDA and A� on cell viability. (A) Representative gel of two inde-pendent A� samples prepared from synthetic A�1–42 as described in Materials andmethods (MS, molecular weight standard). (B) Cells were incubated with 0.5 or 1 �MA� in culture medium for 6 h and immediately assessed for cell viability. Alterna-tively, 1 �M A�-treated cells returned to recovery period of 18 h in conditioned cellculture medium, as described in material and methods, in order to evaluate delayedtoxicity. (C) Cortical cells were incubated with 10 or 100 �M NMDA for 15 min inNa+ medium and immediately assessed for cell viability. Alternatively, cells returnedto recovery period of 18 h in conditioned cell culture medium in order to evaluatedelayed toxicity. (D) Cells were incubated with 100 �M NMDA for 15 min in theabsence or presence of 10 �M ifenprodil, 50 nM NVP-AAM077 or 10 �M MK-801,followed by a recovery period, as described for (B). Data are the mean ± S.E.M. of3 independent experiments performed in duplicates. Statistical analysis: ***p < 0.01

8 I.L. Ferreira et al. / Cell

irect stimulation in Na+ medium without added MgCl2 and inhe presence of 20 �M glycine (to drive the selective activation ofMDARs), by using a microplate reader Spectrofluorometer GeminiM (Molecular Devices, USA) (340/380 nm excitation wavelength,10 nm emission wavelength). Experiments were performed in thebsence or in the presence of NMDA receptor antagonists MK-80110 �M), ifenprodil (10 �M) or NVP-AA077 (50 nM). Unless indi-ated, all plotted values were normalized for baseline levels. Dataere analyzed by using Excel (Microsoft, Seattle, WA, USA) and

rism (GraphPad Software, San Diego, CA, USA) softwares.

.8. Statistical analysis

Data were expressed as the mean ± S.E.M. of the number ofxperiments indicated in the figure legends. Comparisons amongultiple groups were performed by one-way ANOVA, followed by

ukey’s post hoc test. Student’s t-test was also performed for com-arison between two Gaussian populations, as described in figure

egends. Significance was accepted at p < 0.05.

. Results

.1. Expression of NMDA receptor subunits in cerebral corticalultures

In this study we used cultured rat cerebral cortical cells at–8 DIV to evaluate the role exerted by NMDARs, particularlyhose composed by GluN2A and/or GluN2B subunits, in the reg-lation of Ca2+

i homeostasis. Since the expression of GluN2A- andluN2B-NMDARs depends on the developmental stage of the neu-

ons [37,38], it was critical to first examine whether both subtypesf NMDARs were present in these cells at different time in cul-ure. Our results demonstrate that the GluN1 subunit is alreadyxpressed in E16 embryos (0 days in vitro) and that total expressionevels do not change with maturation time points (Fig. 1A). Totalrotein levels of GluN2A and GluN2B subunits of the NMDA recep-or are highly expressed at 8–15 days in culture (Fig. 1A), a timeear synapse formation. Although the results obtained after 15 DIVere slightly greater, data were not significantly different between

and 15 DIV (Fig. 1A). The presence of GluN2A and GluN2B subunitst the surface of plasma membrane of cortical cells maintained for 8IV was also determined by biotinylation assay and protein expres-

ion levels analyzed by Western Blotting. Our results demonstratehat at this stage both NMDAR subunits are present at the mem-rane surface in cortical cultures (Fig. 1B). Reduced GluN2A surface

evels compared to GluN2B surface levels may be accounted for byhe predominant presence of GluN2A at synapses, whereas GluN2Bppear to be mostly extrasynaptic (e.g. [51,52]).

.2. Analysis of preparation of A and cytotoxicity in corticalultures exposed to NMDA and Aˇ

A� preparation was composed by a large percentage (∼60%)f low-n oligomers (with ∼17 and 24 kDa, corresponding to thessembly of 4 or 6 A� peptides) and monomers (by about 40%).

representative gel shows that fresh A�1–42 preparation arenriched in oligomers and monomers and do not contain fibrilsFig. 2A).

Results depicted in Fig. 2B demonstrate that exposure to 0.5 �M� for 6 h did not affect cell viability, even when evaluated after

he recovery period in the presence of a higher concentration of� (1 �M). Conversely, when neurons were exposed for 15 min to

00 �M, but not 10 �M NMDA, a significant decrease in cell viabil-

ty was observed (Fig. 2C). When the toxic effects were evaluatedfter the recovery period, a significant decrease in cell viability wasbserved for both concentrations tested (Fig. 2C). The toxic effect

significantly different when compared with control conditions (Tukey’s post hoctest).

I.L. Ferreira et al. / Cell Calcium 51 (2012) 95– 106 99

Fig. 3. Antagonist modulation of NMDA-induced Ca2+i rise. Cortical cells were stimulated with 10 �M or 100 �M NMDA in the absence or presence of 10 �M ifenprodil,

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0 nM NVP-AAM077 or 10 �M MK-801. (i) Representative tracings. (ii) Results weefore NMDA addition. Data are the mean ± S.E.M. of 3–14 independent experimeignificantly different when compared with NMDA alone (Tukey’s post hoc test).

xerted by 100 �M NMDA, observed after the recovery period, wasounteracted by the GluN2B subunit antagonist ifenprodil (10 �M),hereas treatment with the GluN2A subunit-preferring antagonistVP-AAM077 did not significantly influence the decrease of corti-al neurons viability. As expected, MK-801 (10 �M) fully preventedhe toxic effect exerted by 100 �M NMDA (Fig. 2D).

.3. Involvement of NMDARs in regulating Ca2+i homeostasis

Cerebral cortical cultures (8 DIV) were stimulated with 10 �Mr 100 �M NMDA in the absence or presence of a NMDA receptorntagonist, namely ifenprodil, NVP-AAM077 or MK-801. Selectivetimulation of NMDARs (with NMDA plus glycine in Mg2+-freeedium) induced a concentration-dependent increase in Ca2+

i lev-ls (Fig. 3). Ifenprodil (10 �M) partially, but significantly, inhibitedhe NMDA-induced Ca2+

i rise caused by 10 �M or 100 �M NMDAy about 38.8% (p < 0.001) and 49.6% (p < 0.001), respectively, sug-esting the involvement of GluN2B subunits in the NMDA-induceda2+ response. Conversely, blockade of GluN2A-NMDARs (Fig. 3),chieved with NVP-AAM077 (50 nM), failed to prevent NMDA-nduced Ca2+

i rise upon stimulation with 10 �M or 100 �M NMDA.nterestingly, GluN2A antagonism by NVP-AAM077 significantlyotentiated Ca2+

i rise induced by 100 �M NMDA (p < 0.05). More-ver, NMDA-induced responses were almost completely blockedn the presence of MK-801 (10 �M), decreasing Ca2+

i levels by8.5% and 91.5% (p < 0.001), as compared with the effect of 10 �Mnd 100 �M NMDA alone (Fig. 3). Our results clearly show thatctivation of NMDARs induces an increase in Ca2+

i levels in aoncentration-dependent manner, and that this effect is mostlyediated by GluN2B-containing NMDARs.

.4. A induces changes in intracellular Ca2+ levels throughMDAR activation

Next, we examined whether activation of NMDARs contributeso A�-induced loss of Ca2+

i homeostasis. For this purpose, wexposed neurons to A� at non-toxic micromolar concentrations

0.5 �M or 1 �M) (Fig. 2B).

In order to assess whether GluN2A/B-NMDARs subunits arenvolved in A� peptide-induced loss of Ca2+

i homeostasis, cere-ral cortical neurons were stimulated with A�, and Ca2+

i levels

tted as the difference between the maximum value achieved and the basal valuerformed in duplicates to quadruplicates. Statistical analysis: *p < 0.05, ***p < 0.001

measured in the absence or presence of NMDAR antagonists.Our results show that, similarly to stimulation with NMDA, A�induced a concentration-dependent increase in intracellular Ca2+

levels (Fig. 4). Stimulation with 0.5 �M A� increased Ca2+i levels

below those achieved by stimulation with a sub-toxic concentra-tion (10 �M) of NMDA (Fig. 4i–ii). Moreover, there was a clear trendto return to baseline values. In contrast, after stimulation with1 �M A� this tendency was not observed, and in fact, Ca2+

i lev-els increased similarly to the stimulation observed with the toxicconcentration of 100 �M NMDA (Fig. 4).

In the presence of ifenprodil (10 �M), A�-induced Ca2+i rise

was again partially, but significantly, reduced by 36.5% or 27.6%(p < 0.05) after stimulation with 0.5 �M or 1 �M A�, respectively.Interestingly, as for 100 �M NMDA, stimulation of cortical cultureswith 1 �M A� in the presence of the GluN2A subunit antagonistNVP-AAM077 (50 nM) produced a significant rise in intracellularCa2+

i, by about 48.3% (p < 0.01) (Fig. 4). Conversely, the NMDARantagonistic effect of MK-801 (10 �M) resulted in a strong inhi-bition of intracellular Ca2+

i levels in response to A� stimulation(Fig. 4).

Because an increase in intracellular Ca2+ levels was observedupon incubation with NMDA or A� in the presence of GluN2Asubunit antagonist (NVP-AAM077), and in order to exclude a pos-sible effect exerted by the antagonist itself, we analyzed the basallevels of intracellular Ca2+ recordings in the absence or in the pres-ence of NVP-AAM077, ifenprodil or MK-801. As shown in Table 1,NVP-AAM077 or ifenprodil alone did not account for any effect inintracellular Ca2+ levels, whereas MK-801 slightly, but significantly,decreased basal Ca2+ levels. This observation implicates basal glu-tamate release and is probably due to the potent inhibitory effectof MK-801, which is independent of NMDAR composition.

3.5. Effect of pre-exposure to A in intracellular Ca2+ responseelicited by NMDA

In order to determine the influence of A� on NMDA-evoked

responses, we then investigated the influence of A� pre-exposureon NMDA-induced Ca2+

i changes. Primary cortical cells were pre-exposed (for 180 s) to A� (0.5 or 1 �M), followed by a subsequentstimulation with NMDA (10 or 100 �M); the involvement of

100 I.L. Ferreira et al. / Cell Calcium 51 (2012) 95– 106

Fig. 4. Influence of NMDAR antagonists in Ca2+i changes induced by direct effects of A�. Cortical neurons were exposed to 0.5 �M or 1 �M A� in the absence or in the presence

of 10 �M ifenprodil, 50 nM NVP-AAM077 or 10 �M MK-801. (i) Representative tracings. (ii) Results were plotted as the difference between the maximum value achievedand the basal value before A� addition. Data are the mean ± S.E.M. of 3–9 independent experiments performed in duplicates to quadruplicates. Statistical analysis: *p < 0.05,* s post

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luN2A and GluN2B-NMDAR subunits was assessed by using NVP-AM077 and ifenprodil, respectively.

Interestingly, when NMDA (10 or 100 �M) stimulation wasecondary to pre-exposure to 0.5 �M A�, NMDA-evoked increasen Ca2+

i was not significantly different from that observed withMDA alone (Fig. 5A), which may account for by the fact that.5 �M A�-evoked Ca2+

i responses were transient. As expected,fenprodil (10 �M) partially decreased the Ca2+

i rise triggered by0 �M NMDA (p < 0.05) or 100 �M NMDA (p < 0.001) following A�0.5 �M) exposure by about 42.8% and 69.1%, respectively (Fig. 5A).fter blockade of GluN2A-NMDARs with NVP-AAM077 (50 nM),a2+

i rise did not differ significantly from that observed in thebsence of the antagonist. Notably, the effect of NVP-AAM077 wasot largely different from that observed upon primary NMDA stim-lation (e.g. in the absence of A�), as shown in Fig. 3.

Conversely, when neurons were pre-exposed to 1 �M A�180 s), the secondary stimulation with 10 and 100 �M NMDA-voked Ca2+

i was significantly reduced by 43.5% (p < 0.01) and 62.8%p < 0.001), respectively, when compared with the effect of a pri-

ary stimulation with the same concentration of NMDA (Fig. 5B),ndicating that at this concentration A� largely interferes withMDARs. Notably, 1 �M A�-induced elevation of Ca2+

i was notransient as observed after exposure to 0.5 �M A�.

Ifenprodil (10 �M) further reduced the secondary stimulationvoked by 10 and 100 �M NMDA-induced Ca2+

i rise by 29.3%

nd 53.4% (p < 0.05), respectively (Fig. 5B). Interestingly, ifenprodilnhibited by the same extent NMDA-evoked Ca2+

i in the absenceFig. 3) or following exposure to 1 �M A� (Fig. 5 B), suggesting aimilar contribution of GluN2B subunits in both conditions.

able 1ffect of NMDA receptor antagonists on intracellular basal Ca2+ levels.

Control Ifenprodil

1.23 ± 0.013 (n = 12) 1.23 ± 0.016 (n = 7)

ortical cells were incubated in the absence (control) or in the presence of 10 �M ifenpndependent experiments performed in duplicates to quadruplicates.*** Statistical analysis: p < 0.001 significantly different when compared to condition in th

hoc test); tp < 0.05 significantly different when compared with A� alone (Student’s

Conversely, the antagonism of GluN2A-NMDARs (Fig. 5B)induced by NVP-AAM077 (50 nM) increased NMDA-induced Ca2+

iby 238% after secondary exposure to 100 �M NMDA (p < 0.001).NVP-AAM077-induced Ca2+

i rise under these conditions was highercompared to the primary stimulation with 100 �M NMDA (Fig. 3),suggesting an important role of GluN2A subunits in controllingCa2+

i increases under stress stimuli.These results indicate that 0.5 �M A� does not greatly affect

Ca2+i rise evoked by NMDA stimulation. In contrast, a higher con-

centration of A� (e.g. 1 �M) largely reduces the Ca2+i response

triggered by NMDA, which is alleviated upon blockade of GluN2Asubunits, supporting the notion that NMDAR subunits may differ-ently modulate Ca2+

i particularly under stress conditions.

3.6. Influence of NMDA pre-exposure on Ca2+i rise evoked by Aˇ

To investigate the hypothesis that A� increase in Ca2+i directly

interfered with NMDARs and thus was affected by prior NMDARstimulation, cortical neurons were pre-exposed (180 s) to NMDA(10 or 100 �M) and subsequently stimulated with A� (0.5 or 1 �M).

Our results show that pre-exposure to NMDA in non-toxic(10 �M) and sub-maximal or toxic (100 �M) concentrations sig-nificantly reduces the increase in Ca2+

i induced by A� (0.5 �Mor 1 �M). Indeed, we observed that 10 �M NMDA reduced 0.5and 1 �M A� A�-evoked Ca2+

i rise by 59.8% (p < 0.01) and 87.2%

(p < 0.001), respectively, when compared with the effect of primarystimulation with the same concentration of the soluble peptide(Fig. 6A). Following pre-exposure to 100 �M NMDA, and similarlyto 10 �M, the increase in Ca2+

i due to secondary exposure to 0.5

NVP-AAM077 MK-801

1.23 ± 0.011 (n = 6) 1.13 ± 0.014*** (n = 4)

rodil, 50 nM NVP-AAM077 or 10 �M MK-801. Data are the mean ± S.E.M. of 4–12

e absence of antagonists (Tukey’s post hoc test).

I.L. Ferreira et al. / Cell Calcium 51 (2012) 95– 106 101

Fig. 5. A� modulation of secondary Ca2+i response elicited by NMDA stimulation. Cortical cells were stimulated with (A) 0.5 �M or (B) 1 �M A� followed by a second

stimulation (after 180 s) with 10 �M or 100 �M NMDA in the absence or in the presence of 10 �M ifenprodil or 50 nM NVP-AAM077. (i) Representative tracings. (ii) Resultswere plotted as the difference between the maximum value achieved and the basal value before NMDA addition. The open bars (NMDA) were re-plotted from Fig. 2 in orderto facilitate graph interpretation. Data are the mean ± S.E.M. of 3–5 independent experiments performed in duplicates to quadruplicates. Statistical analysis: ***p < 0.001significantly different when compared to stimulation with NMDA alone (Tukey’s post hoc test); ttp < 0.01 significantly different when compared with NMDA alone (Student’st the efp ulatio

arwuotcHAo(

-test analysis); #p < 0.05, ###p < 0.001 significantly different when compared with

ost hoc test); tp < 0.05 significantly different when compared with the effect of stim

nd 1 �M A� was reduced by 74.5% (p < 0.01) and 83.9% (p < 0.001),espectively, when compared with the effect of primary stimulationith the same concentration of A� (Fig. 6B). GluN2B-NMDAR sub-nit antagonism achieved by ifenprodil (10 �M) did not affect anyf the previously described responses (Fig. 6). These data indicatehat activation of NMDARs by NMDA (10 �M or 100 �M) almostompletely inhibited the intracellular Ca2+

i rise evoked by A�.

owever, the antagonism of GluN2A-NMDARs induced by NVP-AM077 (50 nM) in these conditions reverted the inhibitory effectf 0.5 or 1 �M A�-induced Ca2+

i rise in cells pre-exposed to 10 �MFig. 6A) or 100 �M (Fig. 6B) NMDA.

fect of stimulation with NMDA following pre-exposure to 0.5 or 1 �M A� (Tukey’sn with NMDA following pre-exposure to 0.5 or 1 �M A� (Student’s t-test analysis).

3.7. Intracellular Ca2+ modifications evoked by simultaneousexposure to A and NMDA

Finally, we analyzed the effects in intracellular Ca2+ levels trig-gered by simultaneous exposure to A� and NMDA. The involvementof specific NMDAR subunits was further determined by using ifen-prodil and NVP-AAM077.

Stimulation of cortical cultures with 0.5 �M A� plus 10 �MNMDA potentiated the increase in Ca2+

i levels by 172% or 50%(p < 0.001), respectively, when compared with those achievedupon stimulation with 0.5 �M A� or 10 �M NMDA alone (Fig. 7A).

102 I.L. Ferreira et al. / Cell Calcium 51 (2012) 95– 106

Fig. 6. NMDA modulation of secondary Ca2+i response elicited by A�. Cortical neurons were stimulated with (A) 10 �M or (B) 100 �M NMDA for 180 s, followed by a

second stimulation with 0.5 �M or 1 �M A� in the absence or in the presence of 10 �M ifenprodil or 50 nM NVP-AAM077. (i) Representative tracings. (ii) Results wereplotted as the difference between the maximum value achieved and the basal value before A� addition. The black bars (A�) were re-plotted from Fig. 3 in order to facilitateg erformc t when1

MN((CNNACnw

nd

raph interpretation. Data are the mean ± S.E.M. of 3–4 independent experiments pompared with the effect of A� alone; ##p < 0.01, ###p < 0.001 significantly differen00 �M NMDA (Tukey’s post hoc test).

oreover, simultaneous exposure to 0.5 �M A� plus 100 �MMDA increased Ca2+

i by 300% when compared to 0.5 �M A�p < 0.001) and by 158% when compared to 100 �M NMDAp < 0.001) alone (Fig. 7A). Ifenprodil (10 �M) partially reduceda2+

i rise triggered by simultaneous addition of 0.5 �M A� andMDA (10 �M or 100 �M) to levels similar to those evoked byMDA alone, suggesting the involvement of GluN2B-NMDARs in� response (Fig. 7A). In the presence of NVP-AAM077 (50 nM),a2+

i rise elicited by 0.5 �M A� plus NMDA (10 �M or 100 �M) wasot significantly different from that observed when the antagonist

as absent.

Interestingly, Ca2+i rise achieved upon stimulation of cortical

eurons with 1 �M A� plus 10 �M NMDA was not significantlyifferent from that achieved upon exposure to 1 �M A� alone.

ed in duplicates to quadruplicates. Statistical analysis: **p < 0.01, ***p < 0.001 when compared with the effect of stimulation with A� following pre-exposure to 10 or

However, Ca2+i was 50% higher (p < 0.001) than that observed

in the presence of 10 �M NMDA only (Fig. 7B). Simultaneousstimulation with 1 �M A� plus 100 �M NMDA enhanced Ca2+

i(Fig. 7B) by 54% higher than that observed in response to 1 �MA� alone, and 41% higher than upon stimulation with 100 �MNMDA alone (p < 0.05). Blockade of GluN2B-NMDARs with ifen-prodil (10 �M) partially decreased Ca2+

i rise triggered by 1 �MA� plus NMDA (10 �M or 100 �M) (Fig. 7B). Again, the oppo-site effect was observed in the presence of NVP-AAM077 (50 nM).Indeed, we observed a significant increase in Ca2+

i levels by

about 136% when the cells were stimulated with 1 �M A� plus10 �M NMDA (p < 0.05) and by 160% after exposure to 1 �M A�plus 100 �M NMDA (p < 0.001) in the presence of NVP-AAM077(Fig. 7B).

I.L. Ferreira et al. / Cell Calcium 51 (2012) 95– 106 103

Fig. 7. Simultaneous exposure to A� oligomers and NMDA potentiate Ca2+i rise. Cortical neurons were simultaneously stimulated with (A) 0.5 or (B) 1 �M A� plus 10 or

100 �M NMDA, in the absence or in the presence of 10 �M ifenprodil and 50 nM NVP-AAM077. (i) Representative tracings. (ii) Results were plotted as the difference betweenthe maximum value achieved and the basal value before simultaneous addition of A� oligomers and NMDA. The open (NMDA) and black (A�) bars were re-plotted fromF . of 3–a A� oc nt whe

soau

igs. 2 and 3 in order to facilitate graph interpretation. Data are the mean ± S.E.Mnalysis: *p < 0.05, **p < 0.01, ***p < 0.001 significantly different when compared withompared with A� plus NMDA (Tukey’s post hoc test); tp < 0.05 significantly differe

Together, these results show that A� and NMDA, when present

imultaneously, potentiate Ca2+

i rise, probably as a result of anveractivation of NMDARs. Moreover, data suggest that GluN2A-nd GluN2B-NMDARs exhibit conflicting roles in mediating thenderlying mechanisms.

6 independent experiments performed in duplicates to quadruplicates. Statisticalr NMDA (Tukey’s post hoc test); #p < 0.05, ###p < 0.001 significantly different whenn compared with A� plus NMDA (Student’s t-test analysis).

4. Discussion

Abnormal homeostasis of Ca2+i has been observed in both

elderly and AD subjects [10,39,40]. Several studies have recentlydemonstrated that A� directly interacts with cell function, either

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04 I.L. Ferreira et al. / Cell

y insertion into the membrane to form a cation-conductingore [41], activation of cell surface receptors coupled to Ca2+

nflux [30] or induction of oxidative stress, leading to deregula-ion of mitochondrial homeostasis [9]. Moreover, A� oligomers,reviously described to be more toxic than monomers or fibrils13,17,42], were also described to disrupt the integrity of bothlasma and intracellular membranes, leading to cell death in ahannel-independent way [42]. A� oligomers were also reportedo co-immunoprecipitate with extracellular domains of the GluN1ubunit, suggesting a direct interaction with NMDA receptors [9].ndeed, these authors have also shown that ADDLs bind to or are inlose proximity to NMDARs, triggering neuronal damage throughMDAR-dependent Ca2+ influx.

Although much attention has been given to the toxic profile of� oligomers, recent findings from Jan and collaborators [43] evi-ence that crude A�42 preparations, consisting of a monomericnd heterogeneous mixture of A�42 oligomers, were more toxichan purified monomeric, protofibrillar fractions, or fibrils in dif-erent cell lines and primary neurons. Moreover, these authorsbserved that selective removal of monomeric A�42 from thesereparations, using insulin-degrading enzyme, reversed the toxi-ity of A�42 protofibrils, demonstrating that A�42 toxicity is notinked to specific prefibrillar aggregate(s) but rather to the ability ofhese species to grow and undergo fibril formation, which dependsn the presence of monomeric A�42. Thus, A�42 neurotoxicity isediated by ongoing nucleated polymerization process rather than

y individual A�42 species [43].Our data show that similarly to NMDA (10–100 �M), exposure

o A� preparation (0.5–1 �M) induces a concentration-dependentise in Ca2+

i levels in cerebral cortical cultures expressing equiva-ent total levels of GluN1 as well as GluN2A and GluN2B subunits,nd also surface levels of both GluN2A and GluN2B subunits. Theseffects were almost completely prevented by the non-selectiveMDAR antagonist MK-801, suggesting an activation of NMDARsy A�. Involvement of GluN2A- and GluN2B-NMDAR subtypes wasurther evaluated through pharmacological blockade by competi-ive antagonists of the NMDAR subunits, namely NVP-AAM077 andfenprodil. Our results indicate that Ca2+

i increase upon stimulationith NMDA or A� is mainly due to GluN2B-NMDARs. Indeed, ifen-rodil was able to partially reduce, but not fully inhibit, the Ca2+

influx induced by either NMDA or A�. This could be attributed tohe fact that ifenprodil only blocks pure GluN1/GluN2B NMDARsy approximately 80% [35,36,44]. Incomplete blockade of GluN2B-ontaining receptors may be due to the fact that other NMDARubunits, namely GluN2D or GluN2C, are present in the cortical cellultures and thus may also contribute to the rise in Ca2+

i. Whensing the higher concentration of A� (1 �M) or NMDA (100 �M),ut not the lower concentrations as the stimulating agent, NVP-AM077 antagonism of GluN2A-NMDARs potentiated Ca2+

i riseompared to control conditions (in the absence of NVP-AAM077).hese observations suggest that GluN2A-NMDARs restrain Ca2+

nflux under particular stress stimuli. Our results are supportedy recent studies where application of GluN2B antagonist, but notluN2A-containing receptor antagonist NVP-AAM077, preventedoth apoptosis and necrosis induced by NMDA, indicating the crit-

cal involvement of GluN1/GluN2B, but not GluN2A-containingMDAR subtypes in mediating neuronal death. These findings arelso in accordance with previous reports showing that excitotoxi-ity is triggered by the selective activation of NMDARs containinghe GluN2B subunit [27,38], whereas GluN2A-containing NMDARsromote survival [27]. Indeed, GluN2A- and GluN2B-NMDARsppear to have opposite roles in regulating Ca2+

i in the pres-

nce of oligomeric A� 1–42. These findings support the concepthat dysregulation of Ca2+

i homeostasis is induced by a possi-le interaction of A� with NMDARs, particularly of the GluN2Bubtype. In contrast, in a recent publication, A� oligomers were

m 51 (2012) 95– 106

shown to directly activate NMDA receptors, particularly thosecomposed of GluN2A subunit heterologously expressed in Xeno-pus laevis oocytes [45], which may be related to the expressionof NMDARs subunits in non-neuronal cells. Indeed, in a previousstudy we showed that fibrillary Abeta 1–40 mediated necrotic celldeath through changes in Ca2+ homeostasis in HEK293 cells selec-tively expressing GluN1/GluN2A subunits, but not GluN1/GluN2Bsubunits [46]. An alternative hypothesis for A�-mediated NMDARactivation was recently described to involve A�-evoked glutamaterelease [47].

In line with the findings reported here, a recent study by Alberdiet al. [48] has shown that A� oligomers induce inward currents,Ca2+

i increase, through a mechanism requiring NMDA and AMPAreceptors activation in both rat cortical neurons and hippocam-pal organotypic slices [48]. Accordingly, it was demonstrated thatin the AD brain and human cortical neurons, excitatory synapsescontaining the GluN2B subunit of the NMDA receptor appear to bethe main sites of oligomer accumulation. A� oligomers co-localizewith synaptic markers, and this effect is counteracted by the NMDAantagonists ifenprodil and memantine. The latter is an uncompet-itive NMDAR antagonist that blocks the ion channel formed byNMDARs [14]. Of note, memantine was shown to improve cogni-tive functions in patients with moderate to severe forms of AD,also preventing oxidative stress and calcium influx produced byA� oligomers in hippocampal neuronal cultures [9].

Further evidence suggesting that A� may interact with NMDARswas given by studying the effects of A� on NMDA-evokedCa2+ responses. We observed that pre-exposure to 1 �M A�(which caused sustained elevation of Ca2+

i), but not 0.5 �M A�,significantly reduced the Ca2+

i response triggered by NMDA. Inter-estingly, ifenprodil inhibited NMDA-evoked Ca2+

i by the sameextent, in the absence or subsequent exposure to 1 �M A�, sug-gesting a similar contribution of GluN2B subunits. Conversely,NVP-AAM077 increased Ca2+

i rise under these conditions whencompared to the primary stimulation with 100 �M NMDA, sug-gesting an important role of GluN2A subunits in controlling Ca2+

iincrease under stress stimuli. In agreement with our findingsfollowing 1 �M A� pre-exposure, A� oligomers application wasshown to mimic a state of partial NMDAR blockade, reducingNMDAR activity and NMDAR-dependent calcium influx [13]. More-over, neurons from a genetic mouse model of AD were found toexpress reduced amounts of surface GluN1 subunit of NMDARs[19], and A� 1–42 was also found to reduce surface expressionof the GluN1 subunit, in both cortical and hippocampal neurons[19–21]. Accordingly, A� can interact with NMDAR either throughAbeta-induced modulation of NMDA binding sites and/or A�-mediated protein conformational changes, leading to decreasedNMDA-evoked responses.

Data from the current study also showed that A�-inducedincrease in Ca2+

i was affected by prior receptor stimulation withNMDA, providing additional evidence that A� directly interfereswith NMDARs. Thus, selective activation of NMDARs inhibitedthe Ca2+

i rise evoked by A�, suggesting that A�-induced Ca2+i

responses require the presence of an unstimulated NMDAR.Furthermore, our results demonstrate that simultaneous expo-

sure to A� and NMDA induces overactivation of NMDARs andpotentiates the increase in Ca2+

i levels when compared to stimula-tion with NMDA or A� alone. Interestingly, this effect was greaterfor 0.5 �M than 1 �M A�, suggesting different molecular targetsites at the extracellular domain of the receptor. In line with dataobserved in the presence of 0.5 �M A�, recent studies have reportedthat treatment with A� oligomers potentiates NMDA-evoked firing

and induces a rapid and transient increase in intracellular Ca2+ lev-els that is blocked by memantine in mature hippocampal neurons[9,49]. Moreover, Ca2+ influx elicited by NMDA (10 �M or 100 �M)plus A� (0.5 �M or 1 �M) in both concentrations used was partially

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I.L. Ferreira et al. / Cell

ue to the activation of GluN2B-NMDARs, as demonstrated by ifen-rodil antagonism. Meanwhile, the rising effect of NVP-AAM077as only observed in the presence of 1 �M A�, again suggesting

hat GluN2A-NMDARs act by controlling Ca2+ influx, particularlynder stress stimuli. Consistent results of NVP-AAM077 in the pres-nce of 1 �M A� suggest that GluN2A and GluN2B exert differentialnd opposite roles in regulating Ca2+

i homeostasis. In addition,MDAR-dependent Ca2+ influx triggers a number of intracellularvents that convey the signal downstream to either confer neu-oprotection or to trigger cell death. Thus, GluN2A- and GluN2BMDARs may have antagonist roles in activating pro-survival andro-death signaling pathways. There is a growing body of evi-ence that NMDAR activity has the potential to promote survivalr death in neurons of the central nervous system [50], which maye related to differences in synaptic versus extrasynaptic NMDARignaling. The molecular basis for the apparent differences maye due to distinct locations in GluN2A (synaptic)- and GluN2Bextrasynaptic)-containing NMDARs. As demonstrated by recenteports that focus on NMDAR trafficking, the subunit composi-ion in synaptic versus extrasynaptic membranes depends uponhe phosphorylation state of specific tyrosine residues of GluN2B51]. GluN2A is incorporated into synaptic NMDARs by a mecha-ism involving the cytoplasmic C-terminus [52]. GluN2A may thusecome enriched at synapses, compared to extrasynaptic loca-ions. Therefore, GluN2A-enriched synaptic NMDARs appear toelectively promote survival. Nevertheless, Liu et al. reported thatluN2A-containing NMDARs promote survival, whereas GluN2B-MDARs promote death independent of their location [27]. Inontrast, pro-death NMDAR signaling was reported to be mediatedy GluN2A-NMDARs [53], showing that the subunit composition

s not important in determining excitotoxicity. Indeed, the con-ept that GluN2A partitions near exclusively into synaptic locationsas been challenged recently by findings that GluN2A can endp at extrasynaptic locations in cultured neurons [54] and thathe subunit composition of synaptic and extrasynaptic NMDARs isimilar in 3 week-old acutely dissociated hippocampal slices [55].oreover, it was recently shown that in developing hippocampal

eurons, GluN2B-NMDARs are capable of mediating antagonisticignaling to survival or death, as well as synaptic potentiation,epending on the stimulus employed. This indicates that in imma-ure hippocampal neurons, the subunit composition of the NMDAR

ay not account for dichotomous NMDAR signaling [56]. Anotherontributing factor explaining the differences between synapticnd extrasynaptic pools could be the way they are activated: briefaturating activation in the case of synaptic NMDARs, comparedith chronic, low level activation of extrasynaptic NMDARs by

ath application of glutamate. Differences in the properties ofntracellular Ca2+ transients evoked by these different stimuli mayifferentially affect signaling, even if the overall Ca2+ load is similar25,57]. Therefore, with low doses of NMDA, synaptic NMDA recep-ors are activated far more strongly than extrasynaptic receptors,nabling pro-survival synaptic signaling to dominate and activaterotein kinase B (Akt), extracellular regulated kinases 1 and 2 (Erk/2) and cAMP response element-binding (CREB) proteins. The sce-ario is very different with toxic doses of NMDA; action potentialring is suppressed and the Ca2+ influx is attributable to the directctivation of both synaptic and extrasynaptic NMDA receptors.ence, extrasynaptic signaling is dominant and is coupled to CREB

hut-off pathway [57].As such, the question of whether the GluN2 subunits influence

MDARs signal to survival or death (controlling for the amountf Ca2+ that fluxes into cell) remains unresolved and further stud-

es are required. The possible role of other GluN2 subunits, namelyluN2C and GluN2D, also needs to be clarified. GluN2C- or GluN2D-ontaining receptors appear to give rise to ‘low-conductance’penings with a lower sensitivity to extracellular Mg2+, which may

[

m 51 (2012) 95– 106 105

affect the Ca2+ influx generated by synaptic activation of NMDAR[22]. Another subject that can be targeted in future investigationsconcerns the mechanism underlying the GluN2A-induced modula-tion of Ca2+ rise observed in the present study.

In summary, this work demonstrates that A� preparation com-posed by oligomers and monomers disturbs Ca2+ homeostasisthrough NMDAR activation. In this regard, A� was shown to mod-ulate the effects of NMDAR activation. We also show that GluN2Aand GluN2B subtypes of NMDARs have opposing roles in regulatingCa2+

i homeostasis. However, the question of whether activation ofeach of these NMDAR subtypes signals to pro-survival or pro-deathpathways is yet to be answered. Overall, this work contributes tothe understanding of the molecular basis of AD and thus providesinsight into potential therapeutic targets that may be used to pre-vent or ameliorate early neuronal dysfunction in AD, commonlyattributed to A� oligomers.

Conflict of interest

None declared.

Acknowledgments

This work was supported by Fundac ão para a Ciência e a Tec-nologia (FCT), project reference PTDC/SAU-NEU/71675/2006 andby Lundbeck Foundation. The authors thank researchers at theCenter for Neuroscience and Cell Biology, University of Coimbra:Márcio Ribeiro for technical assistance with fluorimetric measure-ments, Dr. Teresa Oliveira for figure editing, and Dr Rosa Resendeand Rui Costa for technical expertise with A� preparation.

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