Endoplasmic reticulum stress occurs downstream of GluN2B subunit of N -methyl-D-aspartate receptor...

Endoplasmic reticulum stress occurs downstream ofGluN2B subunit of N-methyl-D-aspartate receptor in maturehippocampal cultures treated with amyloid-b oligomers

Rui O. Costa,1 Pascale N. Lacor,2 Ildete L. Ferreira,1 RosaResende,1 Yves P. Auberson,3 William L. Klein,2 Catarina R.Oliveira,1,4 Ana C. Rego1,4 and Claudia M. F. Pereira1,4

1CNC-Center for Neuroscience and Cell Biology, University of Coimbra,

Coimbra, Portugal2Neurobiology and Physiology Department, Northwestern University,

Evanston, IL, USA3Novartis Pharma AG, Basel, Switzerland4Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Summary

Alzheimer’s disease (AD) is a progressive neurodegenerative dis-

order affecting both the hippocampus and the cerebral cortex.

Reduced synaptic density that occurs early in the disease process

seems to be partially due to the overactivation of N-methyl-D-

aspartate receptors (NMDARs) leading to excitotoxicity. Recently,

we demonstrated that amyloid-beta oligomers (AbO), the species

implicated in synaptic loss during the initial disease stages, induce

endoplasmic reticulum (ER) stress in cultured neurons. Here, we

investigated whether AbO trigger ER stress by an NMDAR-depen-

dent mechanism leading to neuronal dysfunction and analyzed

the contribution of GluN2A and GluN2B subunits of this glutamate

receptor. Our data revealed that AbO induce ER stress in mature

hippocampal cultures, activating ER stress-associated sensors and

increasing the levels of the ER chaperone GRP78. We also showed

that AbO induce NADPH oxidase (NOX)-mediated superoxide pro-

duction downstream of GluN2B and impairs ER and cytosolic Ca2+

homeostasis. These events precede changes in cell viability and

activation of the ER stress-mediated apoptotic pathway, which

was associated with translocation of the transcription factor

GADD153 ⁄ CHOP to the nucleus and occurred by a caspase-12-

independent mechanism. Significantly, ER stress took place after

AbO interaction with GluN2B subunits. In addition, AbO-induced

ER stress and hippocampal dysfunction were prevented by ifen-

prodil, an antagonist of GluN2B subunits, while the GluN2A

antagonist NVP-AAM077 only slightly attenuated AbO-induced

neurotoxicity. Taken together, our results highlight the role of

GluN2B subunit of NMDARs on ER stress-mediated hippocampal

dysfunction caused by AbO suggesting that it might be a potential

therapeutic target during the early stages of AD.

Key words: ADDLs; Alzheimer’s disease; amyloid-b oligomers;

endoplasmic reticulum; GluN2B; NMDARs.

Introduction

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disor-

der that currently affects almost 35 million people worldwide. It is a

chronic and progressive neurodegenerative illness characterized by mem-

ory deficits and cognitive decline owing to synaptic and neuronal loss in

the hippocampus and cerebral cortex. The abnormal deposition of amy-

loid-beta (Ab) in these brain regions has suggested that this peptide plays

an essential role in AD pathogenesis (Selkoe, 2001). Initially, only Abdeposited in extracellular plaques was assumed to be neurotoxic, but

recent findings suggest that soluble oligomers of Ab (AbO) might be the

culprits in AD pathology (Sakono & Zako, 2010). Indeed, AbO have been

implicated in the impairment of synaptic plasticity and associated memory

dysfunction during early AD stages and severe neuronal degeneration

and dementia that occur during end disease stages (Lambert et al.,

1998).

In vitro and in vivo studies support that synaptic dysfunction and neuro-

nal death occurring in AD are related with endoplasmic reticulum (ER)

stress (Katayama et al., 2004). Besides, reports revealed altered levels of

ER stress markers in the brain of patients with AD (Hoozemans et al.,

2005). Concurrently, in a transgenic mice model of AD, ER stress-related

genes were proven to be differentially regulated during the initial and

intermediate stages of Ab deposition (Selwood et al., 2009). Further-

more, fibrillar Ab was shown to be involved in neuronal ER stress, trigger-

ing depletion of ER Ca2+ stores and activation of an ER-mediated

apoptotic cell death pathway (Ferreiro et al., 2006; Costa et al., 2010).

Recently, it was also demonstrated that AbO can act as a trigger for ER

stress (Resende et al., 2008b; Nishitsuji et al., 2009).

There is growing consensus that the loss of glutamatergic synapses cor-

relates with the onset and severity of memory impairment that precedes

late neurodegeneration in AD (Parameshwaran et al., 2008). Reduced

synaptic density and selective neuronal dysfunction that occurs in the dis-

ease seems to be partially due to the overactivation of NMDARs and the

consequent increase in intracellular Ca2+ leading to excitotoxicity (Kelly &

Ferreira, 2006). AbO were shown to activate NMDARs containing GluN2B

subunits (Ferreira et al., 2012). However, little is known about the relative

contribution of GluN2A and GluN2B NMDAR subunits to AbO-induced

neuronal dysfunction linked to ER stress and apoptotic cell death. Further-

more, it was suggested that AbO increase NADPH oxidase (NOX)-medi-

ated superoxide production through activation of NMDARs (Shelat et al.,

2008) and recent evidences demonstrate that NOX is the main source of

superoxide radicals generated upon NMDAR activation (Brennan et al.,

2009). Indirect interaction of Ab with NMDARs was also demonstrated,

namely with the extracellular domains of GluN1 subunit, because this sub-

unit was found in the AbO-pulled protein complex (De Felice et al., 2007;

Lacor et al., 2007). AbO interaction with NMDARs has been suggested to

lead to synapse damage and loss of hippocampal neurons (Klein, 2006;

Nathalie Lacor, 2007). In both cortical and hippocampal neuronal cul-

tures, Ab was also demonstrated to modify cell surface expression of

GluN1 and GluN2B subunits (Snyder et al., 2005; Lacor et al., 2007).

Excitatory synapses containing the GluN2B subunit of the NMDAR appear

to be principal sites of AbO accumulation which can be counteracted by

NMDAR antagonists (Deshpande et al., 2009). Recently, it was also

Correspondence

Claudia Maria Fragao Pereira, Center for Neuroscience and Cell Biology, Largo

Marques de Pombal, University of Coimbra, 3004-517 Coimbra, Portugal.

Tel.: +35 123 982 0190; fax: +35 123 982 2776; e-mail:claudia.mf.pereira@

gmail.com

Accepted for publication 6 June 2012

ª 2012 The AuthorsAging Cell ª 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland

823

Aging Cell (2012) 11, pp823–833 Doi: 10.1111/j.1474-9726.2012.00848.xAg

ing

Cell

shown that selective antagonists of NMDARs containing the GluN2B

subunit prevent Ab-mediated inhibition of plasticity in the hippocam-

pus in vivo (Hu et al., 2009). Additionally, the uncompetitive NMDARs

antagonist memantine, used to improve cognitive function in moder-

ate ⁄ severe AD patients, inhibits excitotoxicity because of overactiva-

tion of NMDARs (Parsons et al., 2007). Moreover, memantine

prevents AbO-induced synapse loss, oxidative stress and Ca2+ influx

in hippocampal neuronal cultures (De Felice et al., 2007; Lacor et al.,

2007) and antagonizes the in vivo NMDAR-mediated activity in the

hippocampus (Szegedi et al., 2010). Memantine also reduces neu-

rodegeneration in rat hippocampus induced by Ab (Miguel-Hidalgo

et al., 2002), revealing the therapeutic importance of blocking

NMDARs.

Here, we investigated whether AbO trigger ER stress by a NMDAR-

dependent mechanism in mature hippocampal cultures focusing on

the contribution of GluN2A and GluN2B receptor subunits. Our

results show that AbO-induced ER stress and hippocampal neuronal

dysfunction are prevented by antagonists of NMDAR subunits, partic-

ularly by the GluN2B antagonist ifenprodil. ER stress-mediated hippo-

campal dysfunction occurred after the close interaction between AbO

and GluN2B and subsequent NOX-mediated superoxide radical

production.

This study uncovers a novel pathway underlying hippocampal cell

death triggered by AbO, thus contributing to elucidate the molecular

mechanisms involved in the neurotoxic effect of soluble oligomeric Ab,

one of the crucial features in AD pathology.

Results

In this study, we investigated the hypothesis that AbO induce ER stress in

hippocampal cells by a mechanism dependent on the activation of

NMDARs. Furthermore, we discriminated the role of the NMDAR subunits

GluN2A and GluN2B in this process. As a cellular model, we used primary

mature hippocampal cultures with 17–21 days in vitro (DIV) highly

expressing both GluN2A and GluN2B, as recently shown by us (Mota

et al., 2012).

Blockage of extracellular domains of GluN2B subunits

reduces AbO binding at synaptic terminals

To analyze whether AbO can interact with NMDAR subunits, primary

hippocampal cultures were treated with AbO together with antibodies

directed against the extracellular termini of GluN1, GluN2A or GluN2B.

AbO binding to mature hippocampal cells was prevented when the

extracellular termini of GluN1 and GluN2B, but not of GluN2A, was

blocked. Indeed, a statistically significant reduction in AbO puncta den-

sity occurred in cells treated with AbO in the presence of anti-GluN1 or

anti-GluN2B antibodies, whereas antibodies directed against GluN2A

were ineffective (Fig. 1). AbO binding was not affected by anti-GluN2A

or GluN2B antibodies previously pre-adsorbed with their respective con-

trol antigen peptide or by an antibody anti-C terminus of GluN1

(Fig. 1).

AbO-induced ER stress in hippocampal cultures is prevented

by a GluN2B antagonist

Previous studies from our laboratory established that Ab, including AbO,

induces ER stress in primary cultured cortical neurons (Ferreiro et al.,

2006; Resende et al., 2008a,b). Here, we evaluated the ER stress

response in hippocampal cultures challenged with AbO. After 6 h

treatment with 0.5 lM AbO, a significant increase in the levels of

the ER chaperone GRP78 occurred as revealed by western blot (WB)

analysis (Fig. 2A). Similar results were obtained for XBP-1 (Fig. 2B), a

transcription factor whose mRNA is processed by active IRE1a(reviewed in Naidoo, 2009), one of the three main ER stress sensors,

further providing evidences for altered ER function in the presence of

AbO. Additionally, when hippocampal cells were incubated for 6 h

with 2 lM thapsigargin, a widely used ER stress inducer, GRP78 and

XBP-1 levels significantly increased (Fig. 2A–B) reaching levels similar

to those obtained after AbO stimulation.

To investigate the role of NMDAR subunits in ER stress triggered by

AbO in hippocampal cultures, we used the specific antagonists NVP-

AAM077 and ifenprodil, which block GluN2A and GluN2B subunits,

respectively (Reynolds & Miller, 1989; Liu et al., 2004). Results demon-

strated that ifenprodil, and to a lesser extent NVP-AAM077, can prevent

ER stress that occurs after exposure of hippocampal cultures to AbO,

restoring the levels of GRP78 (Fig. 2A) and XBP-1 (Fig. 2B). However, only

ifenprodil was able to significantly protect cells from AbO-induced ER

stress. For control purposes, TCN 201, another specific antagonist for the

GluN2A subunit (Bettini et al., 2010), and memantine, a noncompetitive

antagonist of the NMDA receptors were also tested (Fig. 2A¢). Results

show an inefficient protection of ER stress by the GluN2A antagonist,

confirming that this subunit is not implicated in the ER stress pathway. By

opposition, memantine, a compound approved for the treatment of

moderate to severe AD, prevented the effect of AbO, further supporting

the involvement of the NMDAR-ER stress pathway in Ab-induced hippo-

campal toxicity.

The disturbance of ER Ca2+ homeostasis is one of the early events impli-

cated in the ER stress response (Szegezdi et al., 2009). Therefore, the ER

Ca2+ content was measured in hippocampal cells 3 h after incubation

with AbO (0.5 lM) or thapsigargin (2 lM) through single-cell Ca2+ imag-

ing. Results confirmed that AbO impairs both ER and cytosolic Ca2+

homeostasis, leading to a significant depletion of ER Ca2+ stores (Fig. 2C–

C¢) accompanied by an increase in cytosolic Ca2+ levels (Fig. 2D–D¢). Simi-

lar results were obtained in thapsigargin-treated cells. In agreement with

results obtained when ER stress markers were analyzed, ifenprodil signifi-

cantly inhibited the loss of ER Ca2+ content observed upon AbO treat-

ment. Although NVP-AAM077 exhibited a tendency to prevent ER Ca2+

depletion after AbO incubation, results were not statistically significant

(Fig. 2C–C¢). Concerning cytosolic Ca2+ levels, both NVP-AAM077 and

ifenprodil were shown to protect against the deleterious effects of AbO.

However, the most prominent effect was afforded by ifenprodil, because

pretreated cells with this GluN2B antagonist presented cytosolic Ca2+

levels similar to control values (Fig. 2D–D¢).To better understand the mechanisms underlying AbO-NMDARs inter-

action and ER stress, we determined the activity of the membrane-bound

enzymatic complex NOX, one of the main sources of superoxide radical.

Our results showed NOX activation in AbO-treated mature hippocampal

cultures that was significantly prevented by apocynin, a specific inhibitor

for this enzyme. Ifenprodil, but not NVP-AAM077, abolished NOX activa-

tion in AbO-treated hippocampal cultures (Fig. 3A). It was also observed

that the increase in the levels of superoxide induced by AbO was signifi-

cantly prevented by ifenprodil and by apocynin (Fig. 3B). On the other

hand, NVP-AAM077 was not able to inhibit AbO-induced superoxide pro-

duction. These results suggest that AbO-induced NOX-mediated superox-

ide generation occurs through a GluN2B-dependent and GluN2A-

independent pathway. Furthermore, ER Ca2+ depletion is significantly

prevented in the presence of apocynin (Fig. 3C), demonstrating the

involvement of NOX-mediated superoxide production on ER stress caused

by AbO.

Role of NMDA receptors in Ab-induced ER stress, R. O. Costa et al.


824

GluN2B antagonist suppresses compromised cell survival

and activation of the ER stress-mediated apoptotic cell

death pathway induced by AbO

After demonstrating that ER stress was activated in hippocampal

cells by AbO through a GluN2B-dependent mechanism, we next

evaluated its role in cell survival, which was analyzed by the MTT

assay. Upon AbO (0.5 lM) or thapsigargin (2 lM) treatment for 6 h,

a significant decrease in cell survival was observed (Fig. 4), which

was even more evident when AbO treatment was extended for

24 h (data not shown). Ifenprodil was demonstrated to prevent hip-

pocampal cells from the decrease in cell viability induced by AbO

incubation during 6 h (Fig. 4A). Similar results were obtained by

nuclear morphology analysis (Fig. 4B). AbO treatment induced

approximately more 25% of cell death in the cultures, an effect

that was prevented by ifenprodil, but not by the GluN2A antagonist

NVP-AAM077.

Then, we investigated (by WB and immunocytochemistry) the levels

and the subcellular localization of GADD153 ⁄ CHOP, a pro-apoptotic

transcription factor that is an important mediator of ER stress-induced

cell death. Indeed, after an incubation period of 6 h with AbO

(0.5 lM), the total levels of GADD153 ⁄ CHOP were significantly

increased when compared to control condition but were not changed

in the presence of antagonists of GluN2A or GluN2B subunits

(Fig. 5B). However, in AbO-treated hippocampal cells, GADD153 ⁄ -CHOP upregulation was associated with enhanced translocation of

this transcription factor to the nucleus (Fig. 5C) that was abolished in

ifenprodil pretreated cells. Although caspase-12 has been implicated

in AbO-induced apoptosis (Nakagawa et al., 2000), hippocampal

cell death triggered by AbO seems to occur independently of this

ER-resident caspase. While thapsigargin (2 lM) significantly decreased

the levels of pro-caspase-12 (Fig. 5A), these levels were not affected

upon treatment with AbO (0.5 lM) for 6 h. Furthermore, pro-caspase-

12 levels were not different between AbO-treated and untreated

cells, in the absence or in the presence of NMDAR antagonists

(Fig. 5A).

Overall, the results described previously report the neuroprotective

effect accomplished by the blockage of NMDARs, in particular of the

GluN2B subunit, on ER stress-mediated hippocampal dysfunction caused

by AbO.

Fig. 1 Amyloid-beta oligomers (AbO) binding at synaptic terminals in mature hippocampal cultures. Cells were treated for 15 min with 0.5 lM AbO and binding was then

evaluated by confocal microscopy with the NU-1 antibody. Blocking of AbO hot-spot binding was performed by incubating cells with 5 lg mL)1 of antibodies directed to the

extracellular epitopes (nt) of GluN1, GluN2A, or GluN2B prior to AbO treatment. Controls included antibodies directed against the intracellular epitopes (ct) of these subunits

or antibodies pre-adsorbed with the control peptide antigen. Values are the means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01 compared with

AbO-treated cells in the absence of antibodies against N-methyl-D-aspartate receptor subunits.



825

Discussion

Several studies highlight the importance of ER stress during the progres-

sion of amyloid pathology in AD (Ferreiro et al., 2007; Nishitsuji et al.,

2009). We have previously shown that fibrillar Ab induces ER stress and

activates an ER stress-mediated apoptotic pathway with mitochondrial

involvement (Ferreiro et al., 2006, 2008a; Costa et al., 2010). However, it

is now well accepted that other Ab species, specifically AbO, are the main

neurotoxic species involved in AD (Klein, 2006; Haass & Selkoe, 2007;

Sakono & Zako, 2010). Taking these evidences into account, we have

recently demonstrated that the ER stress response is also activated by AbO

treatment in primary cultured cortical neurons (Resende et al., 2008a,b).

A strong correlation between AbO levels and the extent of synaptic

damage and cognitive impairment has been demonstrated in AD (Klein

et al., 2001; Nathalie Lacor, 2007). Furthermore, several evidences sup-

port that synaptic targeting by AbO and resulting synaptic alterations are

involved in memory loss in early stages of the disease (Lacor et al., 2004;

Klein, 2006; Lacor et al., 2007) and correlates with binding and activation

of NMDARs in mature hippocampal neurons (De Felice et al., 2007; Lacor

et al., 2007). In these cells, AbO was shown to bind, or to be in close

proximity, to the obligatory GluN1 subunit, leading to neuronal damage

(De Felice et al., 2007). In this work, we address whether AbO cause ER

stress in hippocampal cultures by a mechanism involving the NMDAR,

particularly the GluN2A and GluN2B subunits, described to be enriched at

(A) (A′)

(C) (D)

(C′) (D′)

(B)

Fig. 2 Effect of the GluN2A and GluN2B subunits of the N-methyl-D-aspartate receptors (NMDARs) on amyloid-beta oligomers (AbO)-induced endoplasmic reticulum (ER)

stress and ER vs. cytosolic Ca2 levels in hippocampal cells. Hippocampal cultures were treated during 3 or 6 h with AbO (0.5 lM) for WB or Ca2+ measurements, respectively,

in the absence or in the presence of NVP-AAM077 (50 nM) or ifenprodil (10 lM). For control purposes, TCN 201 (50 lM) another specific antagonist for GluN2A subunit, and

memantine (5 lM), a noncompetitive antagonist of NMDARs were also tested (A¢). Similarly, cells were treated with the widely used ER stressor thapsigargin (2 lM). Total

protein lysates were prepared and analyzed by WB using anti-GRP78 (A, A¢) or anti-XBP-1 (B) antibodies. Levels of Ca2+ in ER stores (C, C¢) and in the cytosol (D, D¢) were

evaluated by monitoring the fluorescence of Fura-2. Initial Fura-2 fluorescence values were used to calculate cytosolic Ca2+ levels and the difference between Fura-2

fluorescence before and after the full depletion of ER Ca2+ content was used to evaluate ER Ca2+ levels. Values are the means ± SEM of at least three independent

experiments. GAPDH was used for WB loading control purposes, and results were normalized to control values. *P < 0.05; **P < 0.01, ***P < 0.001, compared with control

condition. #P < 0.05, ###P < 0.001; significantly different when compared to AbO-treated condition.



826

synaptic and extrasynaptic domains, respectively (Gladding & Raymond,

2011). First, we evaluated the induction of ER stress in AbO-treated pri-

mary cultures of hippocampal cells. Then, we investigated the role of

GluN2A and GluN2B subunits of NMDAR on AbO-induced ER stress and

neurotoxicity using NVP-AAM077 and ifenprodil to antagonize GluN2A

and GluN2B, respectively. As a positive control, we used the known ER

stress inducer thapsigargin.

Under ER stress conditions, cells tend to recover by activating the

unfolded protein response (UPR). However, under severe or prolonged ER

stress, these adaptive signaling pathways fail, leading to activation of

apoptotic cell death (reviewed in Kim et al., 2008). During UPR, the

expression of ER-resident chaperones is up-regulated and can be used to

monitor ER stress. Among these chaperones, GRP78 is considered a valu-

able ER stress marker. Moreover, GRP78 has been shown to be increased

in AD brains (Hoozemans et al., 2005) and to be up-regulated in cultured

neurons upon fibrillar or oligomeric Ab treatment (Ferreiro et al., 2006;

Resende et al., 2008a). Under ER stress conditions, the ER stress sensor

IRE-1a is activated increasing the specific splicing of XBP-1 mRNA. Subse-

quently, XBP-1 induces the expression of chaperones and other proteins

involved in protein folding and degradation. Results presented here show

increased levels of GRP78 (Fig. 2A) and XBP-1 (Fig. 2B) in hippocampal

cells treated with AbO, strongly suggesting that AbO induce ER stress in

these cells.

Disturbance of ER Ca2+ homeostasis is commonly associated with ER

stress (Szegezdi et al., 2009). Previous reports demonstrated that Ab,

including AbO, depletes ER Ca2+ content in cortical neurons, increasing

cytosolic Ca2+ levels and finally leading to apoptosis (Ferreiro et al.,

2008b; Resende et al., 2008b). Furthermore, ER Ca2+ dyshomeostasis is

also implicated in the communication between ER and mitochondria,

acting as a pro-apoptotic signal (Costa et al., 2010). Here, we depict the

early impairment of ER Ca2+ homeostasis in mature hippocampal cells in

the presence of AbO, compared with what occurs in thapsigargin-treated

cells. In fact, a significant depletion of ER Ca2+ content occurred 3 h after

cell treatment (Fig. 2C–C¢), accompanied by an increase in cytosolic Ca2+

levels (Fig. 2D–D¢).When ER stress is too severe, apoptotic cell death is induced through

the activation of the transcription growth arrest factor and DNA damage

gene GADD153 ⁄ CHOP. Our results confirmed the activation of this path-

way by AbO because enhanced protein levels as well as nuclear localiza-

tion of this pro-apoptotic transcription factor was observed upon

treatment with AbO during 6 h, as revealed by WB analysis and fluores-

cence microscopy (Fig. 5B,C). The translocation of GADD153 ⁄ CHOP to

the nucleus was associated with a significant decrease in cell survival

(Fig. 4).

Interestingly, despite the ER-resident caspase-12 has been implicated in

cell death upon Ab exposure (Nakagawa et al., 2000), we did not find

evidences for the conversion of the ER membrane-localized procaspase-

12 into its cytosolic active form in AbO-treated hippocampal cultures

(Fig. 5A). These results suggest that AbO-induced ER stress in hippo-

campal cells occurs by a caspase-12-independent process. Accordingly,

Obeng and Boise (2005) reported that ER stress activates caspase-9

leading to apoptotic cell death by a mechanism that does not require

caspase-12, which is in agreement with the present results.

Besides demonstrating that AbO induces ER stress in hippocampal cells,

we also focused on the possible role of NMDARs. A current therapeutic

approach to slow disease progression in moderate ⁄ severe AD patients

involves the use of memantine, an uncompetitive antagonist of NMDARs.

Memantine, which has been shown to improve cognitive function in

(A) (B)

(C)

Fig. 3 Effect of the GluN2A and GluN2B subunits of N-methyl-D-aspartate receptors (NMDARs) on amyloid-beta oligomers (AbO)-induced NADPH oxidase activation,

superoxide production and endoplasmic reticulum (ER) Ca2+ content in hippocampal cells. Hippocampal cultures were incubated for 6 h with AbO (0.5 lM), in the absence or

in the presence of NVP-AAM077 (50 nM) or ifenprodil (10 lM). The NOX inhibitor apocynin (1 mM) was added at the time of NOX activity assay or 15 min prior to the AbO

stimulus. (A) NOX activity was evaluated by monitoring the chemiluminescence of the probe lucigenin. (B) Superoxide levels were measured using the fluorescent

dihydroethidium (DHE) dye. (C) Levels of Ca2+ in ER stores were accessed by monitoring the fluorescence of Fura-2 in the absence of external Ca2+. The difference between

Fura-2 fluorescence ratio 340 ⁄ 380 nm before and after the addition of thapsigargin (2.5 lM) was used to evaluate ER Ca2+ levels. Values are the means ± SEM of at least

three independent experiments. **P < 0.01; ***P < 0.001, significantly different compared with control values. #P < 0.05, ##P < 0.01, ###P < 0.001; significantly different

when compared to AbO-treated cells.



827

patients with AD, is able to re-establish the glutamatergic system homeo-

stasis (Parsons et al., 2007) and to prevent oxidative stress and Ca2+ influx

induced by AbO in hippocampal neuronal cultures (De Felice et al.,

2007), as well as Ab-induced synaptotoxicity (Lacor et al., 2007) and neu-

rodegeneration (Miguel-Hidalgo et al., 2002). In vivo, memantine was

also shown to reduce the neurodegenerative process in rat hippocampus

(Szegedi et al., 2010), revealing the therapeutic importance of blocking

this receptor. NMDARs activation by AbO is associated with synaptic dys-

function preceding neurodegeneration (Kelly & Ferreira, 2006), suggest-

ing that NMDARs activation also occurs in early phases of AD, when AbO

assume a great importance. Furthermore, AbO were demonstrated to

modify cell surface expression of NMDARs, in particular of the GluN1 and

GluN2B subunits (Snyder et al., 2005; Lacor et al., 2007).

Memantine was able to protect from the AbO toxicity at ER (Fig. 2A¢).Pre-incubation with GluN2A and GluN2B antagonists previous to the

AbO stimulus, especially with the GluN2B antagonist ifenprodil, signifi-

cantly prevented ER stress, cytosolic and ER Ca2+ dyshomeostasis, super-

oxide production, as well as cell death. Indeed, in the presence of

ifenprodil, GRP78, and XBP-1 (Fig. 2A–B) levels remained similar to

controls and were significantly different from those determined in AbO-

treated cells. NVP-AAM077 also presented a protective effect but it did

not reach statistical significance, a result confirmed by the usage of TCN

201, a second antagonist for this subunit (Fig 2A¢), demonstrating the

selectivity of this mechanism toward GluN2B subunits. When Ca2+

homeostasis was evaluated, ifenprodil significantly prevented the deple-

tion of ER Ca2+ content (Fig. 2C–C¢) and the increase in cytosolic Ca2+

levels (Fig. 2D–D¢) induced by AbO. On the other hand, NVP-AAM077

only slightly decreased cytosolic Ca2+ rise (Fig. 2D–D¢). These results sug-

gest that both GluN2A and GluN2B subunits are involved in Ca2+ entry

and subsequent increase in cytosolic levels upon NMDAR activation trig-

gered by AbO but that only GluN2B subunits are implicated in the loss of

ER Ca2+ homeostasis. An increase in NOX activity was also observed in

AbO-treated hippocampal cultures, which was prevented by GluN2B

blockage (Fig. 3A). NOX activation was demonstrated to be implicated in

AbO-induced superoxide production because it was abolished in the pres-

ence of apocynin, a NOX inhibitor (Fig. 3B). Moreover, ifenprodil signifi-

cantly prevented superoxide production in AbO-treated hippocampal

cultures (Fig. 3B) demonstrating the involvement of GluN2B subunit.

These results are in agreement with previous studies demonstrating NOX

as the main source of superoxide upon NMDARs activation (Brennan

et al., 2009) and in cultured neurons exposed to either oligomeric Ab or

NMDA (Shelat et al., 2008). Interestingly, NOX activation was shown to

be involved in the depletion of ER Ca2+ stores in mature hippocampal cul-

tures treated with AbO (Fig. 3C). Nevertheless, the depletion of ER Ca2+

stores in AbO-treated hippocampal cultures can also be explained by the

mechanism of Ca2+-induced Ca2+ release (CICR) in which cytosolic Ca2+

can sensitize the ER Ca2+ release channels IP3R or RyR (Berridge et al.,

2000). The fact that NVP-AAM077 prevented the increase in cytosolic

Ca2+ levels without significant effects on ER Ca2+ depletion, suggests that

the Ca2+ threshold level required for CICR activation is still reached in the

presence of NVP-AAM077, allowing AbO-induced ER Ca2+ release and

depletion of its content. This is an early event that could trigger several

mechanisms culminating in acute cell damage. In fact, our results high-

light the loss of cellular viability and increased cell death by AbO that was

prevented by the GluN2B subunit antagonist ifenprodil (Fig. 4). The pro-

tein levels of the pro-apoptotic transcription factor GAD153 ⁄ CHOP

increased in hippocampal cultures upon AbO exposure, but it was not sig-

nificantly prevented when NMDARs were blocked with ifenprodil

(Fig. 5B). However, the nuclear translocation of GAD153 ⁄ CHOP induced

by AbO was partially attenuated in the presence of this GluN2B antago-

nist (Fig. 5C).

Amelioration of AbO-induced ER stress by ifenprodil in mature hippo-

campal cultures is well correlated with our previous findings showing a

decrease in cell surface levels of GluN2B subunit upon AbO treatment, in

the absence of changes in total protein levels, which we believe to be

associated with its activation followed by internalization (Lacor et al.,

2007). Accordingly, other studies demonstrated that AbO can alter the

subcellular localization of NMDAR subunits, namely of GluN1 (Snyder

et al., 2005; Lacor et al., 2007). Furthermore, we observed a selective

increase in total GluN2A levels, attributed to de novo protein synthesis

without changes in its plasma membrane localization (data not shown)

that might explain the fact that NVP-AAM077 only exerted a slight pro-

tective effect in AbO-treated hippocampal cultures. Concordantly with

the present findings, our previous work showed a preferential role of

GluN2B subunits for AbO-induced Ca2+ rise in cortical neurons (Ferreira

et al., 2012). The relevance of GluN2B subunit for AbO-induced toxicity

in hippocampal cultures was also proven using an antibody competition

assay. Pre-incubation of cells with antibodies against the extracellular epi-

topes of GluN1 and GluN2B subunits decreased AbO binding, whereas

the GluN2A antibody was not effective (Fig. 1). Similar results have been

reported for GluN1 (De Felice et al., 2007) as well as for other synaptic

receptors, such as group 1 metabotropic glutamate receptor 5 (mGluR5;

Renner et al., 2010) and cellular prion protein (PrPC; Lauren et al., 2009),

(A)

(B)

Fig. 4 Role of GluN2A and GluN2B subunits of N-methyl-D-aspartate receptors

(NMDARs) on amyloid-beta oligomers (AbO)-induced loss of hippocampal cell

survival. Hippocampal cells were incubated for 6 or 24 h with AbO (0.5 lM), in the

absence or in the presence of NVP-AAM077 (50 nM) or ifenprodil (10 lM).

Thapsigargin (2 lM) treatment during 6 or 24 h was used as a positive control for

endoplasmic reticulum (ER) stress. Cell viability in treated cells was evaluated by

MTT assay (A) and nuclear morphology analysis using Hoechst 33342 staining

(B), after a 6 or 24 h treatment, respectively. Results are the means ± SEM of values

corresponding at least to three independent experiments, performed in duplicate.

*P < 0.05; **P < 0.01; ***P < 0.001; significantly different with respect to control

values; #P < 0.05, significantly different when compared to AbO-treated cells.



828

suggesting that AbO-binding sites are part of a multiprotein complex.

Furthermore, the localization of the GluN1 ⁄ GluN2B and GluN1 ⁄ GluN2A

complexes have been shown to be different, with predominant localiza-

tion of GluN1 ⁄ GluN2B at perisynaptic sites, outside of the postsynaptic

density (Zhang & Diamond, 2006), which may be the AbO-binding site.

Similar reduction in AbO binding is also afforded by blocking mGluR5,

another glutamate receptor located on the outside ring of the postsynap-

tic density (Lujan et al., 1997). However, so far it has been difficult to

determine which synaptic receptor could serve as a binding site for AbO

as neither antibody competition nor selective receptors knock-out (e.g.,

PrP or mGluR5) have fully eliminated AbO binding (Lauren et al., 2009;

Renner et al., 2010).

In conclusion, this study provides a novel perspective about the interac-

tion and neurotoxicity of AbO on hippocampal cells, as summarized in

Fig. 6. AbO was demonstrated to trigger ER stress by a NMDAR-depen-

dent mechanism. Moreover, it was possible to discriminate the role of the

GluN2A and GluN2B NMDAR subunits, proving that GluN2B has an

important role in AbO-induced hippocampal ER stress and neurotoxicity

that occurs after NMDAR ⁄ GluN2B-AbO interaction. Finally, NOX-medi-

ated superoxide production downstream of GluN2B-AbO interaction was

shown to be involved in the loss of ER Ca2+ homeostasis. These results

suggest that GluN2B subunits may be a potential target for therapeutic

intervention in early stages of AD progression.

Experimental procedures

Preparation of primary hippocampal cell cultures

Hippocampal cell cultures were prepared from hippocampi of E18-E19

Wistar rat embryos. Briefly, the hippocampi were dissected under magni-

fying glass observation and collected in Ca2+- and Mg2+-free Hank’s bal-

anced salt solution (HBSS, in mM): 137 NaCl, 5.36 KCl, 0.44 KH2PO4, 0.34

Na2HPO42H2O, 4.16 NaHCO3, 5 glucose, 1 sodium piruvate, 10 HEPES

(pH 7.4), supplemented with 0.001% phenol red. Tissues were then

treated with 0.035% trypsin (0.4 mg mL)1) for 5 min at 37 �C, and

further washed with type II-S trypsin inhibitor (1.5 mg mL)1 in HBSS).

(A) (B)

(C)

Fig. 5 Role of GluN2A and GluN2B subunits of N-methyl-D-aspartate receptors (NMDARs) on endoplasmic reticulum (ER) stress-mediated apoptotic cell death pathway

triggered by amyloid-beta oligomers (AbO) in hippocampal cells. Hippocampal cells were treated for 6 h with AbO (0.5 lM), in the absence or in the presence of NVP-

AAM077 (50 nM) or ifenprodil (10 lM). In parallel, cells were treated for 6 h with the widely used ER stress inducer thapsigargin (2 lM). Levels of pro-caspase-12 (A) or

GADD153 ⁄ CHOP (B) were analyzed by WB in total protein lysates. (C) GADD153 ⁄ CHOP subcellular localization was evaluated by fluorescence microscopy in cells colabeled

with an anti-GADD153 ⁄ CHOP antibody (green) and Hoechst 33342 (blue), using a 400· magnification. Arrows indicate the absence of nuclear localization of

GADD153 ⁄ CHOP. Values are the means ± SEM of at least three independent experiments, normalized to control. GAPDH was used as loading control. *P < 0.05;

***P < 0.001; significantly different when compared to control conditions.



829

Hippocampi were rapidly rinsed with HBSS and then mechanically dissoci-

ated in Neurobasal Medium (GIBCO BRL; Life Technologies, Paisley, UK),

supplemented with B27 (GIBCO BRL; Life Technologies), 25 lM gluta-

mate, 0.5 mM L-glutamine, 100 U mL)1 penicillin and 100 U mL)1 strep-

tomycin (GIBCO BRL; Life Technologies). Cells were plated on poly-L-

lysine (0.1 g L)1)-coated at a density of 0.045 · 106 cells cm)2 for WB,

0.075 · 106 cells cm)2 for the MTT or at a density of 0.025 · 106

cells cm)2 on glass coverslips, for single-cell Ca2+ imaging or immunocy-

tochemistry. The cultures were maintained at 37 �C in a humidified atmo-

sphere containing 95% air and 5% CO2 for 17–21 days before

treatments. Once a week, half medium was changed with fresh medium

without added glutamate. All animal experiments were carried accord-

ingly to the principles and procedures outlined in the European Union

(EU) guidelines (86 ⁄ 609 ⁄ EEC).

Treatments

Amyloid-beta oligomers were prepared from synthetic Ab1-42 (Bachem,

Bubendorf, Switzerland), according to the procedure first described by

Lambert et al. (1998) with some modifications (Resende et al., 2008b).

Primary mature hippocampal cultures were treated with AbO (0.5 lM,

concentration Ab monomer-equivalent), in the absence or in the presence

of the antagonists of GluN2A or GluN2B subunits of NMDAR, respectively,

NVP-[(R)-[(S)-1-(4-bromophenyl)-ethylamino]-(2,3-dioxo-1,2,3,4 tetrahy-

droquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077; Auberson

et al., 2002) or ifenprodil (Sigma Chemical Co., St. Louis, MO, USA), at

37 �C during 3, 6 or 24 h. When used, NVP-AAM077 (50 nM), and

ifenprodil (10 lM) were added 15 min before AbO treatment. As a posi-

tive control for ER stress, neurons were also treated with the SERCA-AT-

Pase inhibitor thapsigargin (2 lM; Sigma Chemical Co.) for 3, 6, or 24 h.

Western blotting analysis of ER stress markers

Total cell extracts were prepared from treated or untreated cells that were

lysed in a buffer containing (in mM): 25 HEPES-Na, 2 MgCl2, 1 EDTA, 1

EGTA, supplemented with 100 lM PMSF, 2 mM DTT, and a protease

inhibitor cocktail (1 lg mL)1 leupeptin, pepstatin A, chymostatin, and

antipain; Sigma Chemical Co.). The cellular suspension was rapidly fro-

zen ⁄ thawed three times in liquid N2 and centrifuged at 14 000 g, during

5 min. Protein concentration in the supernatant was measured using the

Bio-Rad protein assay reagent (Bio-Rad, Munich, Germany). Samples

were denaturated at 95 �C for 3 min in a six times concentrated sample

buffer: 500 mM Tris, 600 mM DTT, 10% (w ⁄ v) SDS, 30% (v ⁄ v) glycerol

and 0.012% (w ⁄ v) bromophenol blue. Equal amounts of each protein

sample were separated by electrophoresis on a 10% SDS-polyacrylamide

gels (SDS-PAGE) and electroblotted onto PVDF membranes (Amersham

Pharmacia Biotech, Buckinghamshire, UK). The identification of proteins

of interest was facilitated by the usage of a prestained precision protein

standard (Bio-Rad, Hercules, CA, USA). After the proteins were electro-

phoretically transferred, the membranes were blocked for 1 h at room

temperature (RT) in Tris-buffer: 150 mM NaCl, 25 mM Tris–HCl (pH 7.6)

with 0.1% (v ⁄ v) Tween 20 (TBS-T) containing 5% (w ⁄ v) nonfat dry milk

to eliminate nonspecific binding and were next incubated overnight at

4 �C with one of the following primary antibodies, in TBS-T containing

5% (w ⁄ v) nonfat dry milk: mouse polyclonal anti-GRP78 (1:250; BD

Pharmigen, San Diego, CA, USA), mouse monoclonal anti-GADD153 ⁄ -CHOP (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit

polyclonal anti-X-box binding protein-1 (XBP-1, 1:1000; Abcam,

Cambridge, UK), rabbit polyclonal anti-pro-caspase-12 (1:1000; BD

Pharmigen). Mouse monoclonal primary antibody anti-glyceraldehyde-3-

phosphate dehydrogenase (GAPDH, 1:2500; Santa Cruz Biotechnology)

or rabbit polyclonal anti-a-tubulin antibody (1:20 000; Cell Signaling,

Denvers, MA, USA) were used as protein loading controls.

Membranes were washed several times and then incubated in TBS-T

with 1% (w ⁄ v) nonfat dry milk for 2 h at RT with alkaline phosphatase-

conjugated anti-mouse or anti-rabbit secondary antibody at a dilution of

1:20 000 (Amersham Pharmacia Biotech). Immunoreactive bands were

detected on a Bio-Rad Versa Doc 3000 Imaging System after incubation

of membranes with ECF reagent (Amersham Pharmacia Biotech) for

5–10 min.

AbO binding and antibody competition assay

Cells were incubated for 15 min with either 0.5 lM AbO (Lacor et al.,

2004) or the same amount of vehicle solution added directly into the cul-

ture medium. Cells were rinsed twice with neurobasal medium supple-

mented with B27 and fixed for immunocytochemistry experiments as

described previously (Lacor et al., 2004, 2007). Fixed neurons were

blocked with 10% (v ⁄ v) normal goat serum in PBS for 45 min followed by

incubation during 4 h with NU-1 antibody (1:1000; Lambert et al.,

2007). AbO detection was obtained using an Alexa-488 conjugated anti-

mouse secondary antibody for 1 h. Preparations were mounted with Pro-

long mounting medium and imaged using a Leica TCS SP2 Laser confocal

microscope. Blocking of AbO hot-spot binding was performed by incu-

bating cells for 15 min with 5 lg mL)1 antibodies directed to extracellu-

lar epitopes (nt) of GluN1 (NMDAf1), GluN2A (NMDAe1) or GluN2B

(NMDA e2) subunits of NMDAR (from Santa Cruz Biotechnology) prior to

Fig. 6 Summary of the N-methyl-D-aspartate receptor (NMDAR)-dependent

mechanism proposed for amyloid-beta oligomers (AbO)-induced endoplasmic

reticulum (ER) stress in mature hippocampal cells. AbO can interact with or close to

the GluN2B subunit of NMDAR, subsequently leading to the early rise of cytosolic

Ca2+ levels. This increment leads to the activation of NOX and consequent

production of superoxide radicals leading to ER Ca2+ depletion. Then, cellular

response to ER stress is activated to re-establish its normal functioning 1 through

activation of ER stress sensors, including IRE-1a and downstream XBP-and

expression of chaperones, such as GRP78. However, when the AbO toxic insult is

prolonged, the ER stress-mediated apoptotic pathway is induced increasing the

levels of the pro-apoptotic transcription factor GADD153 ⁄ CHOP that is

translocated to the nucleus. AbO-induced ER stress occurs mainly by a GluN2B-

dependent mechanism because it can be prevented by ifenprodil, an antagonist of

GluN2B subunits but not by NVP-AAM077, an antagonist of GluN2A subunits of

NMDARs.



830

AbO treatment for further 15 min. Controls included antibodies directed

against the intracellular epitopes (ct) of these receptor subunits (Chem-

icon International, Inc., Temecula CA, USA) or antibodies pre-adsorbed

with the control peptide antigen. Analysis and quantification of AbO hot-

spots density (number of puncta per length of dendrite) was made using

metamorph software (Molecular Devices Inc., Sunnyvale, CA, USA).

Immunocytochemical detection of the ER stress marker

GADD153 ⁄ CHOP and cell viability evaluation by nuclear

morphology analysis using Hoechst 33342

Treated or untreated cells were rinsed three times with PBS and then

fixed with 3.7% (w ⁄ v) formaldehyde in neurobasal medium (1:1 volume)

for 10 min followed by an additional 10 min undiluted fixative. Cells

were then rinsed extensively in PBS. Coverslip-plated hippocampal cells

were incubated in blocking solution containing 3% (w ⁄ v) BSA in PBS

with 0.1% (v ⁄ v) Triton X-100 for 45 min at RT. Subsequently, cells were

incubated overnight at 4 �C with a mouse monoclonal anti-GADD153 ⁄ -CHOP antibody (1:200; Santa Cruz Biotechnology), diluted in blocking

solution. Thereafter, coverslips were rinsed in PBS with 1% (w ⁄ v) BSA

and incubated for 90 min at RT with the appropriated Alexa-conju-

gated secondary antibody (Molecular probes, Leiden, the Netherlands)

anti-mouse IgG labeled with Alexa Fluor 488 (1:500) diluted in PBS

with 1% (w ⁄ v) BSA. Following additional rinses in PBS, cells were

stained with 10 lg mL)1 Hoechst 33342 (Molecular Probes, Leiden,

the Netherlands) in the dark for 5 min, at RT. Finally, the prepara-

tions were mounted using DakoCytomation fluorescent mounting

medium (DakoCytomation, Carpinteria, CA, USA). Images were col-

lected using an Axiovert 200 M fluorescence microscope (Zeiss, Thorn-

wood, NY, USA) with a 40· objective.

The blue fluorescent Hoechst 33342 dye is a cell-permeable nucleic

acid stainer that allows the distinction between viable and non-viable cells

through the observation of nuclear morphology. Approximately, 300 cells

per condition were examined by blinded counting. Cells showing irregu-

lar, relatively high blue fluorescence or even fragmented nuclei were con-

sidered nonviable. Data were expressed as the percentage of nonviable

cells vs. the total cells counted.

Measurement of ER Ca2+ content

Ca2+ release from ER was evaluated by single-cell Ca2+ imaging using

the fluorescent probe Fura-2 ⁄ AM (Molecular Probes, Eugene, OR,

USA), performed as previously described (Ferreiro et al., 2008b), with

some minor modifications. Treated and untreated cells plated in glass

coverslips were washed two times in Krebs buffer (in mM): 132 NaCl, 4

KCl, 1.4 MgCl2, 6 Glucose, 10 HEPES, 10 NaHCO3, 1 CaCl2, supple-

mented with 0.05 EGTA, pH 7.4. Then, cells were loaded with Fura-

2 ⁄ AM (5 lM) in Krebs buffer supplemented with 0.2% (w ⁄ v) pluronic

acid for 40 min, at 37 �C in the dark. Afterward, cells were washed

three times in Ca2+ -free Krebs buffer and the coverslip was assembled

to perfusion chamber, in the same Ca2+ -free buffer, in an inverted flu-

orescence microscope Axiovert 200 (Zeiss, Jena, Germany). Cells were

alternately excited at 340 and 380 nm using a Lambda DG4 apparatus

(Sutter Instruments Co., Nocato, CA, USA), and emitted fluorescence

was collected and was driven to a coll SNAP digital camera (Roper Sci-

entific, Trenton, NJ, USA). Acquired values were processed using the

MetaFluor software (Universal Imaging Corporation, Buckinghamshire,

UK). Experiments were run for 12 min, and complete ER Ca2+ deple-

tion was accomplished by the addition of thapsigargin (2.5 lM) after a

baseline was established (which was used to determine cytosolic Ca2+

levels). The peak amplitude of Fura-2 fluorescence (ratio at

340 ⁄ 380 nm) was used to evaluate ER Ca2+ content. The fluorescence

increase is proportional to ER Ca2+ content and the absence of external

Ca2+ guarantees that this increase is due solely to the release of Ca2+

from the ER.

Alternatively, plated cells were washed two times in Krebs buffer and

then loaded with Fura-2 ⁄ AM (5 lM) in the same buffer supplemented

with 0.2% (w ⁄ v) pluronic acid for 40 min, at 37 �C in the dark. After-

ward, cells were gently washed three times in Ca2+ -free Krebs medium

and then alternately excited at 340 and 380 nm and the fluorescence

was measured fluorimetrically using a SpectraMax Gemini microplate

reader (Molecular Devices Inc.). ER Ca2+ content was analyzed as

described for single-cell experiments upon addition of thapsigargin

(2.5 lM).

Analysis of NADPH oxidase activity

NADPH oxidase activity was evaluated using the chemiluminescent probe

lucigenin (Sigma Chemical Co.). Briefly, untreated or treated cells were

washed two times with PBS and lysed with 25 mM Tris–HCl (pH 7.4).

Then, 30 lg of protein was added to a 96-well plate containing 100 lM

NADPH and 5 lM lucigenin in a final volume of 200 lL (adjusted with

25 mM Tris–HCl). Luminescence was recorded during 15 min using a

Lmax II 384 microplate reader (Molecular Devices Inc.) and results were

calculated as relative luminescence units (RLU) per min per mg protein

and expressed relatively to control values.

Measurement of intracellular superoxide radical levels

The levels of superoxide anion were assessed using the fluorescent

probe dihydroethidium (DHE; Molecular Probes, Eugene, OR, USA).

Treated or untreated cells were rinsed once with PBS and incubated

with DHE (10 lM) in Krebs medium for 1 h at 37 �C in the dark. Then,

DHE fluorescence was measured for 1 h at 518 nm excitation and

605 nm emission using a SpectraMax Gemini microplate reader (Molec-

ular Devices Inc.). Superoxide levels were determined calculating the

difference between final and basal fluorescence. Results were normal-

ized to control values.

MTT reduction assay to evaluate cell viability

Cell survival was evaluated measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) reduction by metabolic active cells

(Ferreiro et al., 2006). Briefly, untreated or treated cells were incubated

with 0.5 mg mL)1 MTT (Sigma Chemical Co.) prepared in Krebs med-

ium containing (in mM) 132 NaCl, 4 KCl, 1 CaCl2, 1.4 MgCl2, 1.2

NaH2PO4, 6 glucose, and 10 HEPES-Na (pH 7.4) for 2 h at 37 �C. Then,

the medium was removed and the blue formazan crystals were dis-

solved with 0.04 M HCl in isopropanol and quantified at 570 nm in a

microplate reader (Spectra Max Plus 384; Molecular Devices Inc.).

Results were expressed as the percentage (%) of the absorbance mea-

sured in control cells.

Statistical analysis

Results are expressed as means ± SEM of the number of experiments

indicated in the figure captions. Statistical significance was performed

using an analysis of variance (ANOVA), followed by Dunnett’s post hoc

tests for multiple comparisons or by the unpaired two-tailed Student’s

t-test. A P < 0.05 value was considered statistically significant.



831

Acknowledgments

This work was supported by Fundacao para a Ciencia e a Tecnologia

(project PTDC ⁄ SAU-NEU ⁄ 71675 ⁄ 2006) and Lundbeck Foundation.

Rui O. Costa was a recipient of a PhD fellowship from FCT

(SFRH ⁄ BD ⁄ 28403 ⁄ 2006).

Author contributions

Conceived and designed the experiments: Costa RO, Lacor PN,

Oliveira CR, Rego AC, Pereira CF. Performed the experiments: Costa

RO, Lacor PN. Analyzed the data: Costa RO, Lacor PN. Contributed

reagents ⁄ materials ⁄ analysis tools: Rego AC, Ferreira IL, Resende R,

Pereira CF, Auberson YP, Klein WL. Wrote the paper: Costa RO,

Pereira CF.

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Endoplasmic reticulum stress occurs downstream of GluN2B subunit of N -methyl-D-aspartate receptor...

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