Endoplasmic reticulum Ca2+ release through ryanodine and IP3 receptors contributes to neuronal excitotoxicity

G

Y

Ec

Aa

b

c

a

ARAA

KCEEMERI

1

Rcttc

olmraoebgn

Vf

0d

ARTICLE IN PRESSModel

CECA-1104; No. of Pages 9

Cell Calcium xxx (2009) xxx–xxx

Contents lists available at ScienceDirect

Cell Calcium

journa l homepage: www.e lsev ier .com/ locate /ceca

ndoplasmic reticulum Ca2+ release through ryanodine and IP3 receptorsontributes to neuronal excitotoxicity

sier Ruiza,b, Carlos Matutea,b,c, Elena Alberdia,b,c,∗

Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), SpainDepartamento de Neurociencias, Universidad del País Vasco, E-48940 Leioa, SpainNeurotek, Parque Tecnológico de Bizkaia, E-48170 Zamudio, Spain

r t i c l e i n f o

rticle history:eceived 20 July 2009ccepted 20 August 2009vailable online xxx

eywords:alciumxcitotoxicity

a b s t r a c t

Overactivation of ionotropic glutamate receptors induces a Ca2+ overload into the cytoplasm that leadsneurons to excitotoxic death, a process that has been linked to several neurodegenerative disorders. Whilethe role of mitochondria and its involvement in excitotoxicity have been widely studied, the contribu-tion of endoplasmic reticulum (ER), another crucial intracellular store in maintaining Ca2+ homeostasis,is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine(RyR) and IP3 (IP3R) receptors to a neuronal in vitro model of excitotoxicity. NMDA induced a dose-dependent neuronal death, which was significantly decreased by ER-Ca2+ release inhibitors in cortical

ndoplasmic reticulumitochondria

R stressyanodine receptors

P3 receptors

neurons as well as in organotypic slices. Furthermore, ryanodine and 2APB, RyR and IP3R inhibitors respec-tively, attenuated NMDA-triggered intracellular Ca2+ increase and oxidative stress, whereas 2APB reducedmitochondrial membrane depolarization and caspase-3 cleavage. Consistent with ER-Ca2+ homeostasisdisruption, we observed that NMDA-induced ER stress, characterized here by eIF2� phosphorylation andover-expression of GRP chaperones which were regulated by ER-Ca2+ release inhibitors. These results

leasespec

demonstrate that Ca2+ redysfunction and inducing

. Introduction

Excessive activation of ionotropic glutamate receptors (NMDA-, AMPA-R and Kainate-R) induces a massive Ca2+ influx into theell which can trigger neuronal death in the central nervous sys-em (CNS). There is strong evidence suggesting the involvement ofhis glutamate excitotoxicity in acute injury to the CNS and manyhronic neurodegenerative disorders [1,2].

Mitochondria are crucially involved in intracellular Ca2+ home-stasis and accumulation of Ca2+ induced by an excitotoxic stimuluseads to dysfunction of this organelle, including mitochondrial

embrane depolarization, increased production of oxygen freeadicals and release of pro-apoptotic factor cytochrome c andctivation of caspase-3 [3,4]. However, the contribution of otherrganelles such as endoplasmic reticulum (ER) to neurodegen-

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+

excitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

rative processes like Alzheimer’s and Parkinson’s diseases hasecome evident [5] and some studies show that excitotoxicity trig-ers a ER-Ca2+ homeostasis disruption which can contribute toeuronal cell death itself [6,7].

∗ Corresponding author at: Departamento de Neurociencias, Universidad del Paísasco, Barrio de Sarriena s/n, 48940-Leioa, Spain. Tel.: +34 94 601 8280;

ax: +34 94 601 3400.E-mail address: [email protected] (E. Alberdi).

143-4160/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2009.08.005

from ER contributes to neuronal death by both promoting mitochondrialific stress and apoptosis pathways during excitotoxicity.

© 2009 Elsevier Ltd. All rights reserved.

ER is an intracellular organelle of fundamental impor-tance, responsible for protein synthesis and their correctpost-translational “folding”, but also serves as a rapidly exchang-ing Ca2+ store and contributes to the cytosolic calcium signallingcascade by releasing Ca2+ mainly through ryanodine (RyR) andIP3 (IP3R) receptors [8]. The RyR family has three isoforms, allexpressed in the brain, and has multiple allosteric Ca2+ binding sitesresponsible for triggering Ca2+-induced Ca2+ release (CICR) to thecytosol [9]. IP3Rs (isoforms I, II and III) are also expressed in thebrain and activated by Ins(1,4,5)P3, a metabolic product of phos-pholipase C (PLC) activity, but also regulated by IP3-independentpathways [10–13]. These RyRs/IP3Rs play a central role in cell sur-vival, but it has been shown that these receptors can also be crucialin apoptotic cell death [14].

There are two main pathways through which ER can induceapoptosis. One is mitochondria-dependent, consisting on a Ca2+

overload from ER to this organelle. It has been shown that ERand mitochondria are physically and functionally coupled bymicrodomains which involve RyRs and IP3Rs [14–17] and thatCa2+ signalling between these organelles can induce an apopto-

release through ryanodine and IP3 receptors contributes to neuronal

sis crosstalk followed by the mitochondria-specific toxicity eventsmentioned before [18–20]. The other pathway for ER to induceapoptosis is intrinsic and activated by impairment of ER function-ing. When unfolded proteins accumulate or ER calcium stores aredepleted, the unfolded protein response (UPR) is activated [5,21].

ING

Y

2 lcium

TsGeotwbdo

tnIrsprd

2

2

wvapn

2

((cpLCLW

2

2

Spmc1Bwt

2

oo[snot2

33258 staining (5 �g/ml, 10 min). Finally, coverslips were washed

ARTICLEModel

CECA-1104; No. of Pages 9

A. Ruiz et al. / Cell Ca

his highly conserved stress response is initiated by some stress-ensor proteins like PERK, which is controlled by the chaperoneRP78 (BiP) and phosphorylates the translation initiation factorIF2� when activated in stress conditions. UPR leads to a shutdownf translation and an over-expression of ER function related pro-eins like GRP chaperones in order to restore ER capacity. However,hen stress is severe and prolonged, ER itself can induce cell death

y activating, among others, genes like gadd153 (CHOP), which isownstream of the phosphorylated eIF2� and a major componentf ER stress-induced apoptosis [22,23].

Excitotoxicity and ER-induced neuronal death have been linkedo several neurodegenerative disorders, but the molecular mecha-isms and Ca2+ dynamics that lead to pathology remain unknown.

n this study, we have investigated the contribution of ER-Ca2+

elease to a neuronal excitotoxicity in vitro model. We demon-trate that the inhibition of ER-Ca2+ release through RyRs and IP3Rsrotects cortical neurons against NMDA-induced excitotoxicity byeducing cytosolic Ca2+ increase and attenuating both mitochon-rial damage and ER stress.

. Materials and methods

.1. Animals

All experiments were conducted under the supervision andith the approval of our internal animal ethics committee (Uni-

ersity of the Basque Country, UPV/EHU). Animals were handled inccordance with the European Communities Council Directive. Allossible efforts were made to minimize animal suffering and theumber of animals used.

.2. Reagents

MK 801 and ryanodine were obtained from Ascent ScientificBristol, UK). Neurobasal medium, B-27 supplement, calcein-AMcalcein acetoxymethyl ester), CM-H2DCFDA and JC-1 were pur-hased from Invitrogen (Barcelona, Spain). NMDA, HBSS, glycine,oly-l-ornithine and xestospongin C were obtained from Sigma (St.ouis, MO, USA), and salubrinal, 2APB, U73122 and U73343 fromalbiochem (Merck Chemicals, Nottingham, UK). Cytotox 96® forDH release quantification was purchased from Promega (Madison,

I, USA).

.3. Cell culture

.3.1. Primary cortical neuronsCortical neurons were obtained from the cortical lobes of E18

prague–Dawley rat embryos according to previously describedrocedures [24,25]. Neurons were resuspended in B27 Neurobasaledium plus 10% FBS and then seeded onto poly-l-ornithine-

oated 48-well plates or glass coverslips (12 mm in diameter) at.5 × 105 cells per well. The medium was replaced by serum-free,27-supplemented Neurobasal medium 24 h later. The culturesere essentially free of astrocytes and microglia and were main-

ained at 37 ◦C and 5% CO2. Cultures were used at 8–9 days in vitro.

.3.2. Organotypic slice culturesCultures were prepared from coronal cerebral sections (400 �m

f thickness) of brains from Sprague–Dawley rat pups (2–3 daysld) [26] using a modification of the method by Plenz and Kitai27]. The brain was separated into two hemispheres and cortex

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+

excitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

liced using a tissue McIllwain chopper (Mickle Laboratory Engi-eering Co. Ltd.). Slices were selected under microscope and platedn Millicell CM culture inserts (Millipore Ibérica, Spain) and main-ained for 3 days in 75% HME 03 (Cell Concept, Berlin, Germany),mM l-glutamine (Sigma, St. Louis, Mo, USA), 25% horse serum

PRESSxxx (2009) xxx–xxx

(Invitrogen, Barcelona, Spain) and 25 mg/ml gentamycin (Sigma, St.Louis, MO, USA) at 37 ◦C, and then shifted in Neurobasal mediumsupplemented with 0.5% B27 supplement (both from Invitrogen,Barcelona, Spain). Slices were used at 6 days after plating.

2.4. Toxicity assays

Cell toxicity assays were performed as described previously[28] with modifications. Neurons were exposed to NMDA in HBSS(free of Ca2+ and Mg2+) containing 2.6 mM CaCl2, 10 mM glu-cose and 10 �M glycine for 10–30 min at 37 ◦C, depending onthe experiment. Antagonists were present before and during theexcitotoxic insult and cell viability was assessed 24 h later usingcalcein-AM or Citotox 96® colorimetric assay. All experiments wereperformed in quadruplicate and the values provided are the nor-malized mean ± S.E.M. of at least three independent experiments.

For organotypic culture toxicity experiments, slices weretreated with NMDA 50 �M for 1 h in the medium described above.Antagonists were present before (30 min) and during the stimulusand toxicity was determined 24 h later by LDH release quantifi-cation using CytoTox 96® assay (Promega, Madison, WI, USA). Allexperiments were performed in duplicate and LDH release valueswere normalized to 4% paraformaldehyde-fixed slide area arbitraryunits. Finally provided values are the normalized mean ± S.E.M. ofat least three independent experiments.

2.5. Intracellular reactive oxygen species and mitochondrialpotential measurements

Neurons were stimulated with NMDA for 10 min in theabsence or presence of antagonists and loaded with 5-(and-6)-chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetylester (CM-H2 DCFDA) for 30 min for the measurement of gener-ated ROS. Calcein-AM (1 �M) was used to quantify the numberof cells within the reading field and fluorescence was measuredas described previously [29]. For quantification of mitochondrialmembrane potential, cells were loaded 30 min after the excitotoxicstimulus with JC-1 dye for 15 min and red/green fluorescence ratiowas measured. All experiments were performed in quadruplicateand the values provided are the normalized mean ± S.E.M. of at leastthree independent experiments.

2.6. Immunocytochemistry

For the IP3Rs and RyRs expression analysis 8 DIV neurons werefixed with 4% paraformaldehyde for 20 min and permeabilized in1% BSA, 1% normal serum, 0.05% Triton X-100 in PBS for 30 min.Then cells were blocked in 10% BSA, 1% normal serum in PBSfor 1 h and incubated first with the anti-MAP2 antibody (1:100,Sigma) for 1 h at room temperature (RT) in 1% BSA, 1% normalserum in PBS. After washing, cells were labeled with Alexa Fluor®

594-conjugated IgG (1:200, Molecular Probes) for 2 h at RT. Neu-rons were washed in PBS and incubated overnight at 4 ◦C withthe primary antibody: anti-IP3R-1 (1:1000, Affinity Bioreagents);anti-IP3R-2 (1:50, Sta. Cruz Biotechnology); anti-IP3R-III (1:1000,Chemicon); anti-RyR-1 (1:1000, Chemicon); anti-RyR-2 (1:1000,Chemicon); anti-RyR-3 (1:50, Sta. Cruz Biotechnology). After wash-ing with PBS Alexa Fluor® 488-conjugated secondary antibody(1:200, Molecular Probes) was added for 1 h followed by Hoechst

release through ryanodine and IP3 receptors contributes to neuronal

in PBS and mounted using glycergel mounting medium (Dako).Controls without a primary antibody showed no staining.

For the analysis of peIF2� increase, anti-peIF2� (1:1000, CellSignalling) was used as primary antibody and the staining wasperformed as above.

ARTICLE IN PRESSG Model

YCECA-1104; No. of Pages 9

A. Ruiz et al. / Cell Calcium xxx (2009) xxx–xxx 3

F n-speR ptorsa odies

2

ibgtATiSaSibicWI�i

2

pMfHbe4Z(I4amri1aS

ig. 1. Expression of RyRs and IP3Rs in cultured cortical neurons. Neurons (neuroyR-I–III and to IP3R-I–III (both in green) immunoreactivity. Colocalization of recend IP3R-I–III are expressed in MAP2+ cortical neurons. Immunolabeling with antib

.7. Western blotting

Cells (4.5 × 105) were washed with PBS, 0.1 M and harvestedn 50 �l of ice-cold electrophoresis sample buffer. Lysates wereoiled for 10 min and separated by 10 or 15% SDS-polyacrilamideel electrophoresis, depending on the experiment. Samples wereransferred overnight to nitrocellulose membrane (Hybond ECL,mersham Biosciences), blocked in 5% skimmed milk, 5% serum inBST and proteins detected by specific primary antibodies in 5% BSAn TBST overnight at 4 ◦C: anti-peIF2� and anti-eIF2� (1:1000, Cellignalling); anti-KDEL (Grp78, Grp94) (1:1000, Stressgen Biore-gents); anti-�-actin (1:5000, Sigma); anti-caspase-3 (1:1000,anta Cruz Biotechnologies). After washing, membranes werencubated with horseradish peroxidase-conjugated secondary anti-odies (1:2000, Sigma) in 5% skimmed milk, 1% normal serum

n TTBS for 2 h RT and developed using enhanced chemiluminis-ence according to the manufacturer’s instructions (Super Signal

est Dura, Pierce, Rockford, IL, USA). Signals were quantified usingmage-J software (NIH, MA, USA) and values were normalized to-actin signal and provided as the mean ± S.E.M. of at least three

ndependent experiments.

.8. Measurement of [Ca2+]i

[Ca2+]i was determined according to the method describedreviously [30]. Neurons were loaded with fura-2 AM (5 �M;olecular Probes, Invitrogen, Barcelona, Spain) in culture medium

or 30 min at 37 ◦C. Cells were washed in HBSS containing 20 mMEPES, pH 7.4, 10 mM glucose, and 2 mM CaCl2 (incubationuffer) for 10 min at RT. Experiments were performed in a cov-rslip chamber continuously perfused with incubation buffer atml/min. The perfusion chamber was mounted on the stage of aeiss (Oberkochen, Germany) inverted epifluorescence microscopeAxiovert 35) equipped with a 150 W xenon lamp PolychromeV (T.I.L.L. Photonics, Martinsried, Germany) and a Plan Neofluar0× oil immersion objective (Zeiss). Cells were visualized withhigh-resolution digital black/white CCD camera (ORCA; Hama-atsu Photonics Iberica) and [Ca2+]i was estimated by the 340/380

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+

excitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

atio method, using a Kd value of 224 nM. At the end of the assay,n situ calibration was performed with the successive addition of0 mM ionomycin and 2 M Tris/50 mM EGTA, pH 8.5. Data werenalyzed with Excel (Microsoft, Seattle, WA) and Prism (GraphPadiftware, San Diego, CA) software.

cific microtubule associated protein (MAP2), red) are stained with antibodies toand MAP2 immunofluorescence is shown in merged images (yellow). RyR-I and IIto RyR-III was absent in MAP2+ neurons. Scale bar: 50 �m.

2.9. Data analysis

All data are expressed as mean ± S.E.M. (n), where n refers to thenumber of cultures assayed. Statistical analysis was carried out withthe Student’s t-test and significance was determined at p < 0.05.

3. Results

3.1. Ryanodine and IP3 receptors are expressed in culturedneurons

Both RyRs and IP3Rs have been widely described in mammals[31–33] although the specific distribution of these receptors alongthe brain remains unclear. In order to confirm the expression of thedifferent RyR and IP3R isoforms in our in vitro cortical model, weperformed double-immunofluorescence labelling for each receptorisoform with the neuronal marker microtubule associated protein2 (MAP2). Double-immunofluorescence staining showed that IP3Risoforms I, II and III are heavily expressed in cultured neurons andcolocalize with MAP2 (Fig. 1). Immunofluorescence and colocaliza-tion in neurons was also positive for the RyR-I and II. In contrast, noimmunolabeling against the RyR-III receptor was observed (Fig. 1).Double immnofluorescence staining revealed that the IP3R and RyRisoform expression mainly occurred along the neuronal body butalso appeared in the axons and dendrites. Together, these resultsindicate that the three isoforms of IP3Rs and RyR-I and II are presentin cortical neurons in culture.

3.2. Blocking of ER-Ca2+ release reduces calcium overload andexcitotoxic cell death in cortical neurons

Previous results have shown that glutamate accumulation leadsto over-stimulation of postsynaptic glutamate receptors, intracel-lular Ca2+ overload and neuronal cell death [34]. Also, it has beendefined that mitochondrial function is a critical factor that deter-mines the mode of neuronal death in excitotoxicity [35]. However,dysregulation of Ca2+ in ER in excitotoxicity still remains unknown.In order to study whether cytosolic Ca2+ overload by NMDA

release through ryanodine and IP3 receptors contributes to neuronal

receptor activation produces Ca2+ release from ER, we measuredintracellular Ca2+ levels [Ca2+]i increase in neurons by NMDA appli-cation in the presence of 2APB (10 �M) a blocker of IP3Rs [36] orryanodine (50 �M), an antagonist of RyRs [9]. In NMDA-stimulatedneurons, [Ca2+]i increased in 198 ± 15 nM (n = 57). Incubation of

ARTICLE ING Model

YCECA-1104; No. of Pages 9

4 A. Ruiz et al. / Cell Calcium

Fig. 2. Blocking of RyRs and IP3Rs reduces Ca2+ overload and excitotoxicity causedby NMDA receptor activation in cultured neurons. (A) Cells loaded with Fura2-AMwere exposed to NMDA 50 �M in the presence of IP3R antagonist 2APB 10 �M orRyR antagonist ryanodine 50 �M. NMDA-induced [Ca2+]i increase was reduced byboth antagonists. Traces represent [Ca2+]i (means ± S.E.M.) of three independentexperiments. (B and C) Primary cortical neurons were exposed (10 min) to increasingconcentrations of NMDA. Cell death was significantly reduced after a pre-incubationwith ryanodine (10 �M, 45 min), 2APB (10 �M, 10 min), U73122 (5 �M, 30 min) andXeC (1 �M, 1 h). Cell viability was determined 24 h later by fluorimetry using thevw

rt1Cc

s(d

ital dye calcein-AM (1 �M) and data represent means ± S.E.M. *p < 0.05, comparedith control (NMDA alone), paired Student’s t-test.

yanodine or 2APB for 4 min before and during NMDA applica-ion reduced the [Ca2+]i increase to 117 ± 15 nM, n = 51 and to15 ± 8 nM, n = 24, respectively (Fig. 2A). These results indicate thata2+ release through RyRs and IP3Rs contributes to NMDA-induced

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+

excitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

alcium overload in neurons.Next, we analyzed whether Ca2+ release from intracellular

tores was involved in excitotoxic cell death. Application of NMDA10–100 �M) to cultured neurons provoked a dose-dependent celleath, which was attenuated by Rya 10 �M, 2APB 10 �M, xestopon-

PRESSxxx (2009) xxx–xxx

gin C (1 �M, an IP3R antagonist) and U73122 (5 �M, phospholipaseC antagonist). Specifically, neurons were protected from mild exci-totoxicity (7.24 ± 0.94%, n = 11 and 14.4 ± 2.4%, n = 24 of cell death atNMDA 10 and 30 �M, respectively) by all used blockers (Fig. 2B andC). In contrast, cell death from severe excitotoxicity (25.3 ± 1.46%,n = 23 at NMDA 100 �M) was only reduced by the IP3R antagonists2APB and Xe C (Fig. 2C). These results indicate that Ca2+ releasefrom RyR and IP3R after NMDA receptor activation takes part inexcitotoxic cell death in neurons.

3.3. Ryanodine and 2APB attenuate NMDA-induced neuronaldeath in organotypic cortical slice cultures

To verify the involvement of calcium-induced calcium release(CICR) in NMDA-excitotoxicity in a more integral preparation,we used cerebral cortex organotypic slices, a region which usesglutamate as a neurotransmitter. Cortical slices were preincu-bated (30 min) with Rya (50 �M), 2APB (10 �M) and MK-801(50 �M) and then treated with NMDA 50 �M for 1 h. NMDA-induced cell death was confirmed by propidium iodide (PI)staining and quantification of LDH release was used for sub-sequent statistical analysis (Fig. 3). NMDA treatment increasedsignificantly the LDH release to a 190 ± 10% compared to con-trol slices (100%, n = 6) and it was reduced to 158 ± 11% and138 ± 25% by ryanodine and 2APB compared to control slices,respectively. The NMDA receptor inhibitor MK-801 protected theslices from death almost completely (106 ± 4%), showing the speci-ficity of NMDA-induced death. Thus, toxicity assays in organotypiccortical slices showed that neurons are vulnerable to NMDAreceptor activation, an effect that was rescued by RyR and IP3Rantagonists.

3.4. Excitotoxic-induced mitochondrial damage is reduced byER-Ca2+ release inhibitors

Previous reports have shown that mitochondria and ER interactphysically and functionally through microdomains which involveRyRs and IP3Rs and have functional relevance in Ca2+ signalling[37,38]. Thus, we decided to investigate the connection betweenER-Ca2+ release and mitochondria dysfunction during excitotoxic-ity by measuring mitochondrial damage and apoptosis markers likereactive oxygen species (ROS), mitochondrial membrane potentialand cleavage of caspase-3 in the presence of ryanodine and 2APB.

First, using DCFDA, a dye that fluoresces when it is oxidizedwithin living cells, we measured ROS generation in NMDA-stimulated (30 �M, 10 min) cortical neurons after 30 min. In theseexperimental conditions, excitotoxic stimulus increased ROS levelsup to 31.5%. These levels were significantly attenuated by ryan-odine (46 ± 18%, n = 4) and ryanodine together with 2APB (44 ± 14%,n = 4), but not by 2APB alone, comparing to NMDA treatment (100%)(Fig. 4A).

We next examined whether mitochondrial depolarization wasaffected by ryanodine and 2APB after an excitotoxic insult. Neuronswere stimulated by NMDA (30–200 �M) for 5–10 min and mito-chondrial membrane potential was measured 45 min later usingthe fluorescent dye JC-1 (88 ± 1.7–78 ± 6.2% vs. untreated cells,100%, n = 17). NMDA (30 �M) induced in neurons a mitochondrialmembrane potential loss up to 89.2 ± 1.6%, which was almost com-pletely blocked by 2APB (99.5 ± 0.7%, n = 4) whereas ryanodine wasineffective (Fig. 4B). When neurons were pre-treated with ryan-odine together with 2APB, mitochondrial depolarization induced

release through ryanodine and IP3 receptors contributes to neuronal

by higher doses of NMDA (100 and 200 �M) was significantlyreduced as well (data not shown).

Finally, since apoptosis is a crucial toxic event in neuronal exci-totoxicity and is dependent on mitochondrial function [35], westudied the activation (cleavage) of caspase-3 in NMDA-stimulated

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+ release through ryanodine and IP3 receptors contributes to neuronalexcitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

ARTICLE IN PRESSG Model

YCECA-1104; No. of Pages 9

A. Ruiz et al. / Cell Calcium xxx (2009) xxx–xxx 5

Fig. 3. Ryanodine and 2APB prevent NMDA-induced neuronal death in cortical organotypic slices. Cultures were incubated with NMDA 50 �M for 1 h in presence or absenceof ryanodine, 2APB and MK801 (30 min preincubation). (A) Slices were labelled with propidium iodide (PI) and fixed 24 h after NMDA stimulus. Photographs show arepresentative field of PI displaying cell death in organotypic culture slices. Scale bar: 500 �m. (B) Ryanodine and 2APB reduce excitotoxicity in organotypic culture. Corticalslices were exposed to NMDA after 30 min pre-incubation of ryanodine, 2APB or MK-801. For quantification of toxicity samples were taken 24 h later from the medium andLDH activity measured. Toxicity was significantly attenuated by all the inhibitors. Data represent normalized means ± S.E.M. of the ratio between LDH activity values andslice area units. #p < 0.001 compared with non-treated slices; *p < 0.05 compared with NMDA-treated slices, paired Student’s t-test.

Fig. 4. Inhibition of ER-Ca2+ release attenuates mitochondrial depolarization, oxidative stress and apoptosis caused by NMDA. (A) NMDA-induced ROS levels in culturedneurons are reduced in the presence of ER-Ca2+ release blockers. Cortical neurons were stimulated with NMDA (30 �M, 10 min) in the presence of ryanodine (10 �M, 45 min),2APB (10 �M, 10 min) or both and ROS levels were quantified using the CM-H2DCFDA probe (30 �M). Data represent means ± S.E.M. of the % of CM-DCFDA/calcein-AM. (B)NMDA induced a dose-dependent mitochondrial membrane depolarization, which is attenuated by ryanodine and 2APB. Cells were treated in the conditions described aboveand mitochondrial membrane potential was measured using JC-1 fluorescent dye 45 min after the excitotoxic stimulus. Data represent normalized means ± S.E.M. of theJC-1 red/green fluorescence ratio. (C) Caspase-3 cleavage is diminished in the presence of 2APB in NMDA-stimulated neurons. Cells were exposed to NMDA (30 �M, 30 min)after preincubation with inhibitors and harvested 4hrs later for the detection of caspase-3 and its cleavage product by western blot. Data represent optical density caspase-3values normalized to �-actin values (n = 3). #p < 0.05, ##p < 0.001 compared with control (untreated cells); *p < 0.05, **p < 0.01 compared with control (NMDA alone), pairedStudent’s t-test.

ARTICLE IN PRESSG Model

YCECA-1104; No. of Pages 9

6 A. Ruiz et al. / Cell Calcium xxx (2009) xxx–xxx

Fig. 5. Inhibition of ER-Ca2+ release attenuates NMDA-induced ER stress response in cultured cortical neurons. (A) NMDA induces an increase in peIF2� levels in corticalneurons. Cultured neurons were stimulated with NMDA (30 �M, 30 min; N30) and thapsigargin (5 �M, 30 min), fixed and labelled with an antibody which specificallyrecognizes the phosphorylated form of eIF2�. Scale bar: 50 �m (B and C) NMDA-induced peIF2� increase is reduced in the presence of ryanodine. Total protein sampleswere extracted after NMDA (30 �M, 30 min) incubation and peIF2� levels were quantified in the presence of ryanodine (10 �M) and 2APB (10 �M) by western blot. Datarepresent optical density peIF2� values normalized to �-actin values (n = 4). (B and D) NMDA-induced over-expression of GRP78 and GRP94 is attenuated in the presenceof ER-Ca2+ release inhibitors. Total protein samples were extracted as above and GRP78 and GRP94 were identified by western blot using an anti-KDEL sequence antibody.D = 5).i t withl red tot

nccadwNr

ma

ata represent optical density values normalized to corresponding �-actin signal (nnhibitor. Cultured neurons were exposed to NMDA (30 �M, 30 min) after treatmenater (n = 4). Data represent normalized means ± S.E.M. #p < 0.05; ##p < 0.01 compa-test.

eurons when ER-Ca2+ release was blocked. NMDA (30 �M, 30 min)leaved the caspase-3, decreasing the inactive form to a 53 ± 9%ompared to control (100%, untreated cells, n = 4) and a cleav-ge product was detected (Fig. 4C). In the presence of 2APB, theecrease of inactive caspase-3 was attenuated to 97 ± 9% of control,hereas the cleavage product was reduced to 31 ± 6% compared to

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+

excitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

MDA (100% of cleavage, n = 3). Ryanodine resulted ineffective ateducing caspase-3 activation.

These results suggest that ER-Ca2+ release through RyRs andainly through IP3Rs contribute to mitochondrial dysfunction and

poptosis during NMDA insults.

(E) NMDA-induced neuronal death is reduced by salubrinal, an ER stress apoptosisSal 100 �M for 30 min. To determine cell death, LDH release was measured 24 hrsnon-treated cells; *p < 0.05; **p < 0.01 compared to NMDA alone; paired Student’s

3.5. Cortical neurons show a RyR and IP3R-dependent ER stressresponse caused by excitotoxicity in vitro

It has been shown that neurons suffer from ER stress duringkainate-mediated excitotoxicity both in vivo and in vitro due to Ca2+

homeostasis disruption [6]. When this occurs, the “unfolded pro-

release through ryanodine and IP3 receptors contributes to neuronal

tein response” (UPR) is activated, general protein synthesis shutsdown and some Ca2+ homeostasis-related proteins like chaperonsare up-regulated. To study whether NMDA induces ER stress incultured cortical neurons and the possible involvement of RyRsand IP3Rs in this response, we analyzed the phosphorylation of

ING

Y

lcium

etsescniWe1ooba

cbtiiusc(sOiC

4

dcnbctdwi

4

lawetntocbihIdhttp

ARTICLEModel

CECA-1104; No. of Pages 9

A. Ruiz et al. / Cell Ca

I2F�, the downstream target of the ER stress-sensor pPERK, andhe up-regulation of chaperones like GRP78 (BiP) and GRP94. NMDAtimulus (30 �M, 30 min) produced a strong phosphorylation ofIF2�, as well as with thapsigargin (5 �M, 30 min), which inducestore depletion by blocking ER-Ca2+ uptake and serves as positiveontrol (Fig. 5A). NMDA triggered a peIF2� increase to 182 ± 40%,= 4, comparing to non-treated cells (100%), which was completely

nhibited by ryanodine (78 ± 13%) but not by 2APB (Fig. 5B and C).e next analyzed the expression of BiP and GRP94 during the same

xcitotoxic insult and found that both were up-regulated, up to84 ± 31% and 254 ± 32% respectively. The NMDA-induced increasef both GRP78 and GRP94 expression was totally abolished by ryan-dine (to 100 ± 15% and 101 ± 17% of control respectively, n = 4) andy 2APB (104 ± 9% and 80 ± 9% of control respectively, n = 6) (Fig. 5Bnd D).

Apart from this adaptative UPR response, prolonged ER stressan also lead to cell death, a mechanism which has been found toe involved in various diseases of the brain [39]. In order to inves-igate the contribution of ER-mediated cell death in our model, wencubated neurons with NMDA in the presence of salubrinal, annhibitor of peIF2� dephosphorylation which was shown to atten-ate ER stress-induced cell death [40]. Neurons pre-treated withalubrinal (100 �M, 30 min) were exposed to NMDA in the sameonditions that previously induced strong eIF2� phosphorylation30 �M, 30 min). Induced toxicity, in terms of LDH release, wasignificantly reduced from 424 ± 39% to 267 ± 55%, n = 4 (Fig. 5E).verall, these results indicate that, in cortical neurons, NMDA

nduces an ER stress response and subsequent cell death involvinga2+ release from intracellular stores.

. Discussion

It has been previously shown that ER depletion causes neuronaleath while inhibition of ER-Ca2+ release reduces neurotoxicity,onsistent with a strong link between ER-Ca2+ homeostasis andeurodegeneration [21]. In the present study, we show that inhi-ition of ER-Ca2+ release after NMDA stimulation reduces cytosolicalcium increase, mitochondrial damage and excitotoxicity in cul-ured cortical neurons and in organotypic slices. Furthermore, weemonstrate that the NMDA insult induces an ER stress response,hich is also attenuated in the presence of RyR/IP3R inhibitors, giv-

ng new evidence about the contribution of the ER to excitotoxicity.

.1. RyR and IP3R expression

Ca2+ release from the ER can be evoked mainly by specific stimu-ation of two receptors residing in the ER membrane: the ryanodinend IP3 receptors (RyR and IP3R). The RyR isoforms (I, II and III), asell as IP3R-I, II and III are widely expressed in brain with differ-

nt tissue and subcellular distribution [41,42]. However, most ofhe previous studies about the expression of these receptors doot distinguish among different isoforms or cell types. Accordingo RyRs, our immunofluorescence experiments showed expressionf RyR-I and RyR-II in primary cultured cortical neurons, which isonsistent with previous studies about the RyRs localization in therain [41]. However, we found very little or no staining for RyR-III

n vitro, although it has been described in murine cortex by in situybridization and western blot [43]. On the other hand, the three

P3R isoforms have been shown to be expressed in rat brain, with

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+

excitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

ifferent tissue and cell type distribution: the IP3R-I is the mostighly expressed isoform in neurons and IP3R-II has been restrictedo glia [42]. We found that the three isoforms are expressed inhe neuronal cultures assayed, a feature which is consistent withrevious results reported earlier for IP3R-I and III expression [42].

PRESSxxx (2009) xxx–xxx 7

4.2. ER-Ca2+ release and excitotoxicity

Once the presence of RyRs and IP3Rs in our neuronal model wastested, we analyzed the contribution of ER-Ca2+ release throughthese receptors to excitotoxic cell death. ER-Ca2+ buffering activityis essential for the maintaining of the intracellular Ca2+ homeostasisand like mitochondria, its dysfunction has been linked to neuronaldeath. However, there is no strong evidence yet about the associa-tion between ER-Ca2+ release and excitotoxicity. Since ER is capableof Ca2+-induced Ca2+ release (CICR), and since that release itself canbe toxic [44], we investigated whether RyRs and IP3Rs, activated byNMDA-induced Ca2+ entry, contribute to neuronal excitotoxicity.First, as CICR is mainly mediated by RyR in neurons, we blockedRyR-Ca2+ release during NMDA receptor stimulation and foundthat Ca2+ overload and excitotoxic neuronal death was significantlyreduced. These results are consistent with previous studies thatshow the involvement of RyR-Ca2+ release in amyloid-beta causedneurotoxicity, in which RyR antagonist dantrolene partially pre-vented the [Ca2+]i increase and apoptosis induced by the peptide[45]. In addition, dantrolene has been shown to protect neuronsagainst kainic acid-induced excitotoxicity in vivo and in vitro [46].

It has been demonstrated that IP3Rs can also be regulated bycytosolic Ca2+ [10–13] and therefore take part in CICR. Thus, wetested the contribution of IP3Rs to CICR release during excitotoxic-ity. Since a fully specific IP3R antagonist is not available yet, we usedxestospongin C and 2APB, which are widely and commonly usedas IP3R inhibitors in neurons [36,47–51]. On the other hand, thePLC inhibitor U73122 was used to inhibit any IP3 generation [52]induced by excitotoxic stimulus, since it was shown that cytoso-lic Ca2+ has a positive feedback effect onto PLC [53]. We foundthat NMDA-induced cytosolic Ca2+ increase and neuronal deathwas reduced in the presence of 2APB. Furthermore, both XeC andU73122 attenuated excitotoxicity as well, what suggests that thesereceptors are involved in the excitotoxicity-induced Ca2+ home-ostasis disruption and subsequent cell death. The results are inagreement with data from other studies that provided evidenceabout the contribution of IP3Rs to CICR in epilepsy [50] and to prionand amyloid-beta peptides neurotoxicity [48]. Consistent with dataobtained from excitotoxicity assays performed in primary culturedneurons, we observed that inhibition of both RyRs and IP3Rs pro-tects neurons against NMDA-excitotoxicity in organotypic corticalcultures. Cultured brain slices are considered a useful tool for neu-robiological research because they preserve neuronal connectivityand glial-neuronal interactions among other features [54].

4.3. Endoplasmic reticulum-mitochondria crosstalk inexcitotoxicity

Ca2+ entry to the cytoplasm and its uptake by mitochondriafollowed by its membrane depolarization plays a major role inneuronal excitotoxicity [55,56]. Furthermore, close associationsbetween mitochondria and ER have been revealed, which couldbe relevant for apoptosis-inducing Ca2+ signalling [16,57]. Indeed,previous studies found an apoptotic crosstalk between the twoorganelles followed by Bcl-2-regulated cytochrome c release andactivation of caspases [58]. More related to our study, it wasrecently shown that RyR and IP3R-mediated Ca2+ release activatesthe mitochondrial apoptotic pathway in amyloid-beta-inducedneurotoxicity [19]. Mitochondrial membrane depolarization andoxidative stress are associated to mitochondria-specific toxicity

release through ryanodine and IP3 receptors contributes to neuronal

pathway, and were all attenuated in the presence of RyR/IP3Rinhibitors. In addition, blocking of IP3Rs by 2APB reduced caspase-3cleavage in NMDA-stimulated neurons, consistent with previ-ous studies which showed mitochondrial transition pore openingdriven by IP3R-Ca2+ release [37]. Thus, these results provide new

ING

Y

8 lcium

en

4

rtsrdcstbrtrlEitioaws

Cndadtsmeat

A

tMd

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

ARTICLEModel

CECA-1104; No. of Pages 9

A. Ruiz et al. / Cell Ca

vidence about a functional link between ER and mitochondria ineuronal damage.

.4. ER stress in neuronal excitotoxicity

Several studies have claimed for a strong link between neu-odegeneration and endoplasmic reticulum stress [5,21]. However,he molecular mechanisms underlying this ER-Ca2+ homeosta-is disruption by excitotoxicity as well as the role of specificelease channels are still unclear. In the present study, we firstemonstrate that NMDA induces an ER stress response in culturedortical neurons. In neurons exposed to NMDA, we observed atrong and fast phosphorylation of the eIF2� and an increase inhe expression of the GRP94 and GRP78 ER-resident chaperones,oth widely described as characteristic events in neuronal injury-elated UPR [6,59–61]. In addition, we show that ER-Ca2+ releasehrough RyRs is involved in this response, since inhibition of theeceptors with ryanodine diminishes both the eIF2� phosphory-ation and GRP78 and 94 expression increase, likely by avoidingR depletion. This data are in agreement with previous exper-ments showing that RyR blocking attenuated GRP78 antisensereatment enhanced apoptosis in neurons [7]. Moreover, NMDA-nduced excitotoxic neuronal death was decreased in the presencef salubrinal, a peIF2� dephosphorylation and ER stress-inducedpoptosis inhibitor, what suggests that ER-specific apoptosis path-ays are involved in cortical neuron excitotoxicity, as previously

hown in kainate stimulated hippocampal neurons [6].In summary, the results reported here provide evidence that ER-

a2+ release through RyRs and IP3Rs contribute to NMDA-inducedeuronal excitotoxicity in vitro. Inhibition of intracellular stores-ependent Ca2+ release during the excitotoxic insult results in anttenuation of cytosolic Ca2+ increase, mitochondrial membraneepolarization, ROS generation and activation of caspase-3. In addi-ion, blocking of ER-Ca2+ release reduced the ER stress response,uggesting that RyR/IP3R-dependent Ca2+ release activates bothitochondrial and ER-specific cell death pathways in neuronal

xcitotoxicity. These data suggest that ER depletion may representn interesting target of therapies for neurodegenerative diseaseshat involve excitotoxic neuronal damage.

cknowledgments

We thank O. López, S. Martín and H. Gómez, for technical assis-ance. This study was supported by CIBERNED and by grants from

inisterio de Educación y Ciencia, Gobierno Vasco and Universidadel País Vasco. A.R. held a fellowship from Gobierno Vasco.

eferences

[1] S.A. Lipton, P.A. Rosenberg, Excitatory amino acids as a final common pathwayfor neurologic disorders, N. Engl. J. Med. 330 (1994) 613–622.

[2] D.W. Choi, Glutamate neurotoxicity and diseases of the nervous system, Neuron1 (1998) 623–634.

[3] A. Atlante, P. Calissano, A. Bobba, S. Giannattasio, E. Marra, S. Passarella, Gluta-mate neurotoxicity, oxidative stress and mitochondria, FEBS Lett. 497 (2001)1–5.

[4] C.M. Luetjens, N.T. Bui, B. Sengpiel, et al., Delayed mitochondrial dysfunctionin excitotoxic neuron death: cytochrome c release and a secondary increase insuperoxide production, J. Neurosci. 20 (2000) 5715–5723.

[5] W. Paschen, T. Mengesdorf, Endoplasmic reticulum stress response and neu-rodegeneration, Cell Calcium 38 (2005) 409–415.

[6] A.L. Sokka, N. Putkonen, G. Mudo, et al., Endoplasmic reticulum stress inhibitionprotects against excitotoxic neuronal injury in the rat brain, J. Neurosci. 27(2007) 901–908.

[7] Z. Yu, H. Luo, W. Fu, M.P. Mattson, The endoplasmic reticulum stress-responsive

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+

excitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

protein GRP78 protects neurons against excitotoxicity and apoptosis: suppres-sion of oxidative stress and stabilization of calcium homeostasis, Exp. Neurol.155 (1999) 302–314.

[8] A. Verkhratsky, O.H. Petersen, The endoplasmic reticulum as an integrating sig-nalling organelle: from neuronal signalling to neuronal death, Eur. J. Pharmacol.447 (2002) 141–154.

[

[

PRESSxxx (2009) xxx–xxx

[9] S. Bardo, M.G. Cavazzini, N. Emptage, The role of the endoplasmic reticulumCa2+ store in the plasticity of central neurons, Trends Pharmacol. Sci. 27 (2006)78–84.

10] J. Yang, S. McBride, D.O. Mak, et al., Identification of a family of calcium sensorsas protein ligands of inositol trisphosphate receptor Ca(2+) release channels,Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 7711–7716.

11] N.N. Kasri, A.M. Holmes, G. Bultynck, et al., Regulation of InsP3 receptor activityby neuronal Ca2+-binding proteins, EMBO J. 23 (2004) 312–321.

12] R.E. Hagar, A.D. Burgstahler, M.H. Nathanson, B.E. Ehrlich, Type III InsP3 recep-tor channel stays open in the presence of increased calcium, Nature 396 (1998)81–84.

13] M.D. Bootman, M.J. Berridge, H.L. Roderick, Activating calcium release throughinositol 1,4,5-trisphosphate receptors without inositol 1,4,5-trisphosphate,Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 7320–7322.

14] G. Hajnoczky, G. Csordas, M. Madesh, P. Pacher, Control of apoptosis by IP(3) andryanodine receptor driven calcium signals, Cell Calcium 28 (2000) 349–363.

15] G. Csordas, C. Renken, P. Varnai, et al., Structural and functional features andsignificance of the physical linkage between ER and mitochondria, J. Cell Biol.174 (2006) 915–921.

16] R. Rizzuto, S. Marchi, M. Bonora, et al., Ca(2+) transfer from the ERto mitochondria: when, how and why, Biochim. Biophys. Acta (2009),doi:10.1016/j.bbabio.2009.03.015.

17] G. Szabadkai, K. Bianchi, P. Varnai, et al., Chaperone-mediated coupling of endo-plasmic reticulum and mitochondrial Ca2+ channels, J. Cell Biol. 175 (2006)901–911.

18] D.G. Breckenridge, M. Germain, J.P. Mathai, M. Nguyen, G.C. Shore, Regula-tion of apoptosis by endoplasmic reticulum pathways, Oncogene 22 (2003)8608–8618.

19] E. Ferreiro, C.R. Oliveira, C.M. Pereira, The release of calcium from the endo-plasmic reticulum induced by amyloid-beta and prion peptides activates themitochondrial apoptotic pathway, Neurobiol. Dis. 30 (2008) 331–342.

20] D.M. Arduino, A.R. Esteves, S.M. Cardoso, C.R. Oliveira, Endoplasmic reticu-lum and mitochondria interplay mediates apoptotic cell death: relevance toParkinson’s disease, Neurochem. Int. 55 (2009) 341–348.

21] A. Verkhratsky, Physiology and pathophysiology of the calcium store in theendoplasmic reticulum of neurons, Physiol. Rev. 85 (2005) 201–279.

22] S.J. Marciniak, D. Ron, Endoplasmic reticulum stress signaling in disease, Phys-iol. Rev. 86 (2006) 1133–1149.

23] S. Oyadomari, M. Mori, Roles of CHOP/GADD153 in endoplasmic reticulumstress, Cell Death Differ. 11 (2004) 381–389.

24] N.S. Cheung, C.J. Pascoe, S.F. Giardina, C.A. John, P.M. Beart, Micromolar l-glutamate induces extensive apoptosis in an apoptotic–necrotic continuum ofinsult-dependent, excitotoxic injury in cultured cortical neurones, Neurophar-macology 37 (1998) 1419–1429.

25] J.A. Larm, N.S. Cheung, P.M. Beart, (S)-5-fluorowillardiine-mediated neuro-toxicity in cultured murine cortical neurones occurs via AMPA and kainatereceptors, Eur. J. Pharmacol. 314 (1996) 249–254.

26] F. Cavaliere, K. Dinkel, K. Reymann, Microglia response and P2 receptor partic-ipation in oxygen/glucose deprivation-induced cortical damage, Neuroscience136 (2005) 615–623.

27] D. Plenz, S.T. Kitai, Organotypic cortex-striatum-mesencephalon cultures: thenigrostriatal pathway, Neurosci. Lett. 209 (1996) 177–180.

28] D. Schubert, H. Kimura, P. Maher, Growth factors and vitamin E modify neuronalglutamate toxicity, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 8264–8267.

29] M.R. Campos-Esparza, M.V. Sanchez-Gomez, C. Matute, Molecular mechanismsof neuroprotection by two natural antioxidant polyphenols, Cell Calcium 45(2009) 358–368.

30] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+ indicators withgreatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440–3450.

31] S.H. De, L. Missiaen, J.B. Parys, et al., Isoform diversity of the inositol trispho-sphate receptor in cell types of mouse origin, Biochem. J. 322 (Pt 2) (1997)575–583.

32] J.J. Mackrill, R.A. Wilcox, A. Miyawaki, K. Mikoshiba, S.R. Nahorski, R.A. Challiss,Stable overexpression of the type-1 inositol 1,4,5-trisphosphate receptor in Lfibroblasts: subcellular distribution and functional consequences, Biochem. J.318 (Pt 3) (1996) 871–878.

33] A.M. Suburo, J. Rodrigo, M.L. Rossi, et al., Immunohistochemical localization ofthe inositol 1,4,5-trisphosphate receptor in the human nervous system, BrainRes. 601 (1993) 193–202.

34] R. Sattler, M. Tymianski, Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death, Mol. Neurobiol. 24 (2001) 107–129.

35] M. Ankarcrona, J.M. Dypbukt, E. Bonfoco, et al., Glutamate-induced neuronaldeath: a succession of necrosis or apoptosis depending on mitochondrial func-tion, Neuron 15 (1995) 961–973.

36] Z. Gu, Q. Jiang, A.K. Fu, N.Y. Ip, Z. Yan, Regulation of NMDA receptors by neureg-ulin signaling in prefrontal cortex, J. Neurosci. 25 (2005) 4974–4984.

37] G. Szalai, R. Krishnamurthy, G. Hajnoczky, Apoptosis driven by IP(3)-linkedmitochondrial calcium signals, EMBO J. 18 (1999) 6349–6361.

38] G. Szalai, G. Csordas, B.M. Hantash, A.P. Thomas, G. Hajnoczky, Calcium signaltransmission between ryanodine receptors and mitochondria, J. Biol. Chem.

release through ryanodine and IP3 receptors contributes to neuronal

275 (2000) 15305–15313.39] W. Paschen, Dependence of vital cell function on endoplasmic reticulum cal-

cium levels: implications for the mechanisms underlying neuronal cell injuryin different pathological states, Cell Calcium 29 (2001) 1–11.

40] M. Boyce, K.F. Bryant, C. Jousse, et al., A selective inhibitor of eIF2alpha dephos-phorylation protects cells from ER stress, Science 307 (2005) 935–939.

ING

Y

lcium

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[injury induces endoplasmic reticulum stress with different cell-type dependentresponse, J. Neurochem. 102 (2007) 1242–1255.

ARTICLEModel

CECA-1104; No. of Pages 9

A. Ruiz et al. / Cell Ca

41] R. Bouchard, R. Pattarini, J.D. Geiger, Presence and functional significance ofpresynaptic ryanodine receptors, Prog. Neurobiol. 69 (2003) 391–418.

42] A.H. Sharp, F.C. Nucifora Jr., O. Blondel, et al., Differential cellular expression ofisoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain,J. Comp. Neurol. 406 (1999) 207–220.

43] G. Giannini, A. Conti, S. Mammarella, M. Scrobogna, V. Sorrentino, The ryan-odine receptor/calcium channel genes are widely and differentially expressedin murine brain and peripheral tissues, J. Cell Biol. 128 (1995) 893–904.

44] Z. Pan, D. Damron, A.L. Nieminen, M.B. Bhat, J. Ma, Depletion of intracellular Ca2+

by caffeine and ryanodine induces apoptosis of Chinese hamster ovary cellstransfected with ryanodine receptor, J. Biol. Chem. 275 (2000) 19978–19984.

45] E. Ferreiro, C.R. Oliveira, C. Pereira, Involvement of endoplasmic reticulum Ca2+

release through ryanodine and inositol 1,4,5-triphosphate receptors in the neu-rotoxic effects induced by the amyloid-beta peptide, J. Neurosci. Res. 76 (2004)872–880.

46] B.O. Popescu, M. Oprica, M. Sajin, et al., Dantrolene protects neurons againstkainic acid induced apoptosis in vitro and in vivo, J. Cell Mol. Med. 6 (2002)565–569.

47] A.E. Chavez, J.H. Singer, J.S. Diamond, Fast neurotransmitter release triggered byCa influx through AMPA-type glutamate receptors, Nature 443 (2006) 705–708.

48] E. Ferreiro, R. Resende, R. Costa, C.R. Oliveira, C.M. Pereira, An endoplasmic-reticulum-specific apoptotic pathway is involved in prion and amyloid-betapeptides neurotoxicity, Neurobiol. Dis. 23 (2006) 669–678.

49] K. Hernandez-Fonseca, L. Massieu, Disruption of endoplasmic reticulum cal-cium stores is involved in neuronal death induced by glycolysis inhibition incultured hippocampal neurons, J. Neurosci. Res. 82 (2005) 196–205.

50] S. Pal, D. Sun, D. Limbrick, A. Rafiq, R.J. DeLorenzo, Epileptogenesis induces long-

Please cite this article in press as: A. Ruiz, et al., Endoplasmic reticulum Ca2+

excitotoxicity, Cell Calcium (2009), doi:10.1016/j.ceca.2009.08.005

term alterations in intracellular calcium release and sequestration mechanismsin the hippocampal neuronal culture model of epilepsy, Cell Calcium 30 (2001)285–296.

51] N. Solovyova, P.R. Moult, B. Milojkovic, J.J. Lambert, J. Harvey, Bi-directionalmodulation of fast inhibitory synaptic transmission by leptin, J. Neurochem.108 (2009) 190–201.

[

PRESSxxx (2009) xxx–xxx 9

52] S.Z. Lin, G.M. Yan, K.E. Koch, S.M. Paul, R.P. Irwin, Mastoparan-induced apopto-sis of cultured cerebellar granule neurons is initiated by calcium release fromintracellular stores, Brain Res. 771 (1997) 184–195.

53] K.W. Young, M.S. Nash, R.A. Challiss, S.R. Nahorski, Role of Ca2+ feedback onsingle cell inositol 1,4,5-trisphosphate oscillations mediated by G-protein-coupled receptors, J. Biol. Chem. 278 (2003) 20753–20760.

54] B.H. Gahwiler, M. Capogna, D. Debanne, R.A. McKinney, S.M. Thompson, Organ-otypic slice cultures: a technique has come of age, Trends Neurosci. 20 (1997)471–477.

55] A.K. Stout, H.M. Raphael, B.I. Kanterewicz, E. Klann, I.J. Reynolds, Glutamate-induced neuron death requires mitochondrial calcium uptake, Nat. Neurosci. 1(1998) 366–373.

56] M.W. Ward, A.C. Rego, B.G. Frenguelli, D.G. Nicholls, Mitochondrial membranepotential and glutamate excitotoxicity in cultured cerebellar granule cells, J.Neurosci. 20 (2000) 7208–7219.

57] G. Csordas, G. Hajnoczky, Sorting of calcium signals at the junctions of endo-plasmic reticulum and mitochondria, Cell Calcium 29 (2001) 249–262.

58] J. Hacki, L. Egger, L. Monney, et al., Apoptotic crosstalk between the endo-plasmic reticulum and mitochondria controlled by Bcl-2, Oncogene 19 (2000)2286–2295.

59] T. Mengesdorf, S. Althausen, I. Oberndorfer, W. Paschen, Response of neuronsto an irreversible inhibition of endoplasmic reticulum Ca(2+)-ATPase: rela-tionship between global protein synthesis and expression and translation ofindividual genes, Biochem. J. 356 (2001) 805–812.

60] C. Penas, M.S. Guzman, E. Verdu, J. Fores, X. Navarro, C. Casas, Spinal cord

release through ryanodine and IP3 receptors contributes to neuronal

61] S. Reijonen, N. Putkonen, A. Norremolle, D. Lindholm, L. Korhonen, Inhibitionof endoplasmic reticulum stress counteracts neuronal cell death and proteinaggregation caused by N-terminal mutant huntingtin proteins, Exp. Cell Res.314 (2008) 950–960.