Amyloid β peptide oligomers directly activate NMDA receptors

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Cell Calcium 49 (2011) 184–190

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Amyloid ␤ peptide oligomers directly activate NMDA receptors Laura Texidó a,b , Mireia Martín-Satué a,b , Elena Alberdi a,c , Carles Solsona a,b , Carlos Matute a,c,∗ a

Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Spain Department of Pathology and Experimental Therapeutics and Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Faculty of Medicine, Universidad de Barcelona, E-08907 Hospitalet de Llobregat, Spain c Departamento de Neurociencias, Universidad del País Vasco, E-48940 Leioa, Spain b

a r t i c l e

i n f o

Article history: Received 2 December 2010 Received in revised form 1 February 2011 Accepted 3 February 2011 Available online 23 February 2011 Keywords: Amyloid ␤ NMDA receptors Xenopus oocytes Neuron death Alzheimerˇıs disease

a b s t r a c t Amyloid beta (A␤) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which A␤ oligomers cause neurotoxicity remain unknown. We recently reported that A␤ oligomers cause intracellular Ca2+ overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether A␤ oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, A␤ oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK801. Intriguingly, the amplitude of the responses to A␤ oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca2+ induced by A␤ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that A␤ oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent A␤ damage to neurons in Alzheimerˇıs disease. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Soluble oligomers of the amyloid-␤ (A␤) peptide are neurotoxins that contribute to Alzheimer’s disease (AD) pathology. A␤ oligomers form in mouse models of AD [1,2] and accumulate in the cerebrospinal fluid (CSF) [3,4] and brain tissue of AD patients [4–6]. The abundance of A␤ oligomers also correlates with disease progression [7–9]. A␤ oligomers exert neurotoxic effects by disrupting the integrity of both plasma and intracellular membranes [10] and by accumulating at excitatory synapses, impairing synapse function [11,12]. A␤ oligomers may be toxic due to calpain activation following Ca2+ influx mediated by NMDA receptors [13] and due to oxidative stress and mitochondrial damage initiated by NMDA receptor activation [14]. Notably, A␤ oligomer neurotoxicity is neutralized by NMDA receptor antagonists, both in dissociated neurons and in organotypic hippocampal cultures [14]. Since A␤ peptides can regulate the release of glutamate [15], the deleterious effects of A␤ oligomers could be caused by overactivation of NMDA receptors due to excessive glutamate at synapses. However, silencing neuronal activity with tetrodotoxin

∗ Corresponding author at: Departamento de Neurociencias, Universidad del País Vasco, E-48940 Leioa, Spain. Tel.: +34 94 601 3244; fax: +34 94 601 3400. E-mail address: [email protected] (C. Matute). 0143-4160/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2011.02.001

and removal of extracellular Ca2+ to minimize transmitter release does not have any effect on the NMDA receptor-mediated currents induced by A␤ oligomers [14]. Because of that observation, we hypothesized that A␤ oligomers may directly activate NMDA receptors, in particular those formed by the NR2A and NR2B subunits which are abundantly expressed in the cerebral cortex and hippocampus. In the current study, we use a Xenopus oocyte expression system to show that A␤ oligomers activate recombinant NMDA receptors formed by NR1 and NR2A or NR2B subunits. These subunits are abundantly expressed in the cerebral cortex and hippocampus, two regions that are particularly vulnerable to AD.

2. Materials and methods 2.1. Animals and solutions Oocytes were obtained from mature Xenopus laevis females (Centre d’Elevage des Xénopes, Montpellier, France). Stage V and VI oocytes were collected and maintained at 16–17 ◦ C in Barth’s solution (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3 )2 , 0.41 mM CaCl2 , 0.82 mM MgSO4 , 2.40 mM NaHCO3 , and 20 mM HEPES at pH 7.5) supplemented with penicillin (100 IU/ml) and streptomycin (0.1 mg/ml).

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2.2. Expression of NMDA receptors comprised of NR1/NR2A and NR1/NR2B subunits The plasmids containing the cDNA for human NR1 (pCIneo), NR2A (HumNR2A-pcDNAI/Amp) and NR2B (HumNR2B6pcDNAI/Amp) subunits were provided by Dr. Paul Whiting, Ph.D., Senior Director, Neuroscience Research Centre (Merck Sharp & Dohme Research Laboratories, Harlow, Essex, UK). Plasmids were linearized with XbaI (NR1) or EcoRV (NR2A, NR2B), and the products were used for cRNA synthesis in vitro using the mCAP RNA Capping Kit (Stratagene). The resulting capped cRNA was aliquoted and stored at −80 ◦ C. The cRNA was injected (50 nl, 1.5 ␮g/␮l) into Xenopus oocytes 48–72 h before electrophysiological recording was performed. The follicular cell layer was removed from the oocytes by enzymatic digestion [incubation of the oocytes with 0.25 mg/ml collagenase type 1A; (Sigma)] 24 h before recording. Control oocytes were injected with 50 nl of ultrapure water and subjected to the same enzymatic digestion procedure. 2.3. Preparation of Aˇ oligomers A␤1-42 (ABX, Radeberg, Germany) was dissolved in hexafluoroisopropanol (Sigma, St Louis, MO, USA) to obtain a 1 mM solution and then aliquoted in sterile microcentrifuge tubes. The hexafluoroisopropanol was removed under vacuum using a SpeedVac and the peptide film was stored (desiccated) at −80 ◦ C. For the aggregation protocol, the peptide was first resuspended in dry DMSO (Sigma, St Louis, MO, USA) to a concentration of 5 mM, then Hams F-12 (PromoCell, Labclinics, Barcelona, Spain) was added to bring the peptide to a final concentration of 100 ␮M and incubated at 4 ◦ C for 24 h. The preparation was centrifuged at 14,000 × g for 10 min at 4 ◦ C to remove insoluble aggregates, and the supernatants containing soluble A␤1–42 were transferred to clean tubes and stored at 4 ◦ C. A␤ peptide aggregation was evaluated by SDS–PAGE electrophoresis and Western blotting using 6E10 antibodies (Covance, Emeryville, California) (Supplementary Fig. S1). 2.4. Electrophysiology All recordings were made at room temperature (20–22 ◦ C) and performed using a two-microelectrode voltage-clamp system (Axoclamp-2A, Axon Instruments, USA). Microelectrodes were filled with KCl (3 M) and had resistances ranging from 1 to 2 M. Membrane currents were low-pass filtered at 10 Hz and recorded on a PC using the Whole Cell Analysis program (WinWCP v3.2.8 and v3.3.3) at twice the rate of the filter frequency. The membrane potential of the oocytes was clamped at −80 mV. Currents due to NMDAR expression were evoked by bath application of a solution containing 100 ␮M l-glutamic acid and 10 ␮M glycine for 25 s. The flow rate was 8 ml/min. Oocytes were exposed to A␤ oligomers that were manually applied to the surface of the cells for 25 s. In some cases, manual delivery induced a change in the slope of the inward current that was considered an artifact. The oligomers were washed out by re-establishing the flow; this, too, sometimes induced a small artifact current. When investigating the effects of NMDAR antagonists, oocytes were preincubated with the NMDA receptor antagonists for 10 min before adding the A␤. The total recording time was 2.5 min. The recording solution was 115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 , and 10 mM HEPES at pH 7.4. 2.5. Calcium imaging in cultured neurons from the cerebral cortex Primary neuron cultures were obtained from the cortical lobes of E18 Sprague–Dawley rat embryos according to previously described procedures [14]. Cells were resuspended in B27 Neu-


robasal medium plus 10% FBS (Sigma, St Louis, MO, USA) and then seeded onto poly-l-ornithine-coated glass coverslips at 1 × 105 cells per coverslip (12 mm in diameter) and 48-well plates at 1.5 × 105 per well. One day later, the medium was replaced by serum-free-, B27-supplemented Neurobasal medium. The cultures were essentially free of astrocytes and microglia; they were maintained at 37 ◦ C and 5% CO2 . Cultures were used 8–10 days after plating. [Ca2+ ]i was determined as previously described [14]. Oligodendrocytes were loaded with fura-2 AM (5 ␮M; Molecular Probes, Invitrogen, Barcelona, Spain) in culture medium for 30 min at 37 ◦ C. Cells were washed in HBSS containing 20 mM HEPES, pH 7.4, 10 mM glucose, and 2 mM CaCl2 (incubation buffer) for 10 min at room temperature. Experiments were performed in a coverslip chamber continuously perfused with incubation buffer at 4 ml/min. The perfusion chamber was mounted on the stage of a inverted epifluorescence microscope (Zeiss Axiovert 35, Oberkochen, Germany) equipped with a 150 W xenon lamp Polychrome IV (T.I.L.L. Photonics, Martinsried, Germany) and a Plan Neofluar 40× oil immersion objective (Zeiss). Cells were visualized with a high-resolution digital black/white CCD camera (ORCA C4742-80-12 AG; Hamamatsu Photonics Iberica). [Ca2+ ]i was estimated by the 340/380 ratio method, using a Kd value of 224 nM. At the end of the assay, in situ calibration was performed with the successive addition of 10 mM ionomycin and 2 M Tris/50 mM EGTA, pH 8.5. Data were analyzed with Excel (Microsoft, Seattle, WA, USA) and Prism (GraphPad Software, San Diego, CA, USA) software. 2.6. Drugs l-Glutamic acid and glycine were obtained from Sigma (St. Louis, MO, USA). The NMDA receptor antagonists D-AP5, MK-801, and memantine were obtained from Tocris Bioscience (Cookson, Bristol, UK). 2.7. Calculations and statistics Statistical analysis was performed using SigmaStat 3.2 software (SPSS Inc., Chicago, IL, USA). Values are reported as the mean ± S.E.M. The Student’s t-test was used to compare the means of two independent groups of normally distributed data. 3. Results 3.1. Effects of Aˇ oligomers on oocytes injected with water (control) We first studied the interaction of soluble A␤ oligomers with NMDARs that were heterologously expressed in X. laevis oocytes. Defolliculated oocytes were injected with water and voltage clamped at −80 mV. To determine the response to NMDAR agonists, l-glutamic acid (100 ␮M) plus glycine (10 ␮M) or glycine (10 ␮M) alone was added. No currents were observed in the water-injected oocytes (Fig. 1A). Similarly, no currents were observed when A␤ oligomers (1 ␮M) were added with glycine (Fig. 1A). 3.2. Aˇ oligomers elicit inward currents in NR1/NR2A-injected oocytes We next measured the effect of A␤ oligomers on NR1/NR2Asubtype NMDARs. When l-glutamic acid and glycine were added to oocytes expressing NR1/NR2 NMDARs, there was a reversible inward current with a mean maximal amplitude of 0.45 ± 0.07 ␮A and a mean electrical charge of 14.70 ± 2.60 ␮C (n = 10) (Fig. 1B). Applied alone, glycine also evoked inward currents with a mean amplitude of 0.16 ± 0.04 ␮A and a mean charge or 4.45 ± 1.14 ␮C


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Fig. 1. Amyloid beta (A␤) oligomers activate NR1/NR2A-subtype NMDARs. (A) Neither co-application of l-glutamic acid and glycine nor application of glycine alone induced inward currents in control oocytes injected with water (negative control). Similarly, no currents were evoked by co-application of A␤ and glycine to water injected-oocytes. (B) l-Glutamic acid and glycine induced reversible inward currents in NR1/NR2A-expressing oocytes (top), as did glycine alone (middle). Currents evoked by glycine were significantly higher when glycine was co-applied with A␤ in NR1/NR2A-expressing oocytes (bottom). (C) Current amplitude and electrical charge evoked by glycine alone (10 ␮M) or by glycine co-applied with A␤ (1 ␮M). Values are shown relative to the values obtained when l-glutamic acid (10 ␮M) and glycine were co-applied (positive control). *p ≤ 0.05, **p ≤ 0.01. The black arrow (↓) indicates an artifact due to flow restoration.

(n = 10) (Fig. 1B). In contrast to the effect observed in water-injected oocytes, when A␤ and glycine were co-applied to NR1/NR2Ainjected oocytes, reversible inward currents were observed with a mean amplitude of 0.32 ± 0.04 ␮A and a mean charge of 9.10 ± 1.60 ␮C (n = 10) (Fig. 1B). This response was greater than that induced by glycine alone. Specifically, the response to lglutamic acid and glycine was considered the positive control (100%); the current amplitude and charge induced by glycine were 28.55 ± 2.50% and 24.70 ± 1.93% of control values, respectively, whereas the current amplitude and charge induced by A␤ oligomers plus glycine were 69.70 ± 15.35% and 57.25 ± 11.90% of control values, respectively (Fig. 1C). 3.3. Aˇ oligomers elicit inward currents in NR1/NR2B-injected oocytes The effect of A␤ oligomers on NR1/NR2B-subtype NMDARs was investigated next using the same oocyte expression system. Co-application of l-glutamic acid and glycine to NR1/NR2Binjected oocytes evoked a reversible inward current with a mean maximal amplitude of 0.09 ± 0.01 ␮A and a mean electrical charge of 2.52 ± 0.40 ␮C (n = 12) (Fig. 2A). Glycine alone evoked a small inward current, 0.01 ± 3.83 × 10−3 ␮A and 0.28 ± 0.13 ␮C (n = 12), whereas glycine co-applied with A␤ oligomers evoked a larger response, 0.03 ± 6.31 × 10−3 ␮A and 0.56 ± 0.12 ␮A (n = 12) (Fig. 2A). Compared to control (addition of l-glutamic acid and glycine), the current amplitude and charge induced by glycine

alone were 15.20 ± 1.80% and 7.13 ± 1.36% of control values, respectively, whereas the current amplitude and charge induced by A␤ oligomers plus glycine was significantly higher, at 48.38 ± 3.85% and 27 ± 2.90% of control values, respectively.

3.4. NMDA receptor antagonists block Aˇ-activated currents in NR1/NR2A and NR1/NR2B-injected oocytes To further characterize the properties of the A␤-induced currents, we measured the effects of NMDA receptor antagonists on current generation in oocytes. Specifically, we measured currents in oocytes that expressed NR1/NR2A or NR1/NR2B receptors and were exposed to A␤ oligomers plus glycine in the presence or absence of D-AP5 (100 ␮M), MK-801 (50 ␮M), or memantine (50 ␮M) (Fig. 3A and C). Compared to the NR1/NR2A-injected oocytes exposed to A␤ oligomers plus glycine in the absence of inhibitor (control; 0.29 ± 0.03 ␮A and 8.44 ± 1.23 ␮C; n = 12) (Fig. 3A), NR1/NR2Ainjected oocytes preincubated with D-AP5 showed currents with a significantly reduced mean amplitude (22 ± 3.16% of control, n = 4) and electrical charge (15.64 ± 4.22% of control, n = 4) (Fig. 3B). Similarly, the amplitude and electrical charge were substantially reduced compared to control when the oocytes were preincubated with MK-801 (current amplitude, 4.45 ± 1.11% of control; current charge, 2.67 ± 0.25% of control; n = 4) or memantine (current amplitude, 7.93 ± 3.94% of control; current charge, 2.20 ± 1.12% of control; n = 4) (Fig. 3B).

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Fig. 2. Amyloid beta (A␤) oligomers activate NR1/NR2B-subtype NMDARs. (A) l-Glutamic acid and glycine, as well as glycine alone, induced reversible inward currents in NR1/NR2B-expressing oocytes. The currents evoked by A␤ together with glycine were significantly higher than those induced by glycine alone. (B) Current amplitude and electrical charge evoked by glycine alone (10 ␮M) or by glycine co-applied with A␤ (1 ␮M). Values are shown relative to the values obtained when l-glutamic acid (10 ␮M) and glycine were co-applied (positive control). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. The black arrow (↑) indicates an artifact due to A␤ delivery.

In the same way, compared to NR1/NR2B-injected oocytes exposed to A␤ oligomers plus glycine in the absence of inhibitor (control; 0.04 ± 9.4 × 10−3 ␮A and 0.82 ± 0.20 ␮C (n = 16) (Fig. 3C), NR1/NR2B-injected oocytes preincubated with D-AP5 showed currents with a significantly reduced mean amplitude (19.90 ± 10.60% of control; n = 4) and electrical charge (28.20 ± 8.60% of control, n = 4) (Fig. 3C). The amplitude and electrical charge were also substantially reduced compared to control when the oocytes were preincubated with MK-801 (current amplitude, 4.19 ± 2.15% of control; current charge, 15.83 ± 3% of control; n = 5) or memantine (current amplitude, 18.91 ± 5.70% of control; current charge, 24.25 ± 5.91% of control; n = 7) (Fig. 3D). These results provide evidence that A␤ oligomers interact directly with NMDARs and activate currents through the receptors. 3.5. Pharmacology of Aˇ-activated responses in cortical neurons We previously observed that A␤-activated responses in cortical neurons were blocked by the wide spectrum NMDA receptor antagonists AP5, MK801 and memantine [14]. Since A␤-activated currents in oocytes expressing NR1/NR2A subunits have a larger amplitude, we next examined whether A␤ oligomers favor the activation of native receptors having that conformation versus those with NR1/NR2B, by using ifenprodil which selectively blocks NMDA receptors with NR2B subunits [16], and calcium imaging. We observed that the increase in the cytosolic Ca2+ concentration after A␤ incubation was abolished by AP5, whereas ifenprodil only reduced 29.2 ± 9% of the response (3 ␮M; n = 64–71 for each condition; Fig. 4). This indicates that native NMDA receptors activated by A␤ oligomers preferably lack NR2B subunits. 4. Discussion The results presented here show that in the absence of glutamate, A␤ oligomers activated recombinant NR1/NR2A and NR1/NR2B receptors that were expressed in Xenopus oocytes. In addition, the kinetics and pharmacology of the responses to A␤ oligomers were comparable to the responses to glutamate.

It is very unlikely that expressed NMDA receptors interact with Xenopus transporters or ion channels in a non-physiological manner. Native oocytes and control oocytes injected with vehicle do not express glutamate transporters and receptors as well as tetrodotoxin-sensitive sodium channels which are only induced after encoding mRNAs are injected [17,18]. Moreover, expression of cloned genes in oocytes does not alter the expression and function of endogenous genes and gene products present in the oocyte membrane [19]. A␤ oligomers interact with cell membranes, form cationconducting pores, and activate surface receptors that are coupled to Ca2+ influx; further, oxidative stress due to A␤ oligomers may lead to dysregulation of mitochondrial homeostasis [11,20–22]. In our experiments, A␤ oligomers did not alter the permeability of the membrane when applied to control (water-injected) oocytes; thus, effects on membrane permeability require the direct interaction of A␤ with the heterologously expressed NMDARs. Consistent with the findings reported here, it has been suggested that A␤ interacts directly with NMDARs and modulates channel properties [11,23]. Thus, application of A␤ oligomers to mature hippocampal neurons potentiates NMDA-evoked firing and induces a rapid and transient increase in intracellular Ca2+ levels that is blocked by memantine [11,24]. In turn, A␤ oligomers induce Ca2+ influx, calpain activation, and dynamin-1 degradation that is mediated by NMDA receptor activation [13]; A␤ oligomers also induce mitochondrial damage that leads to neuronal death [14]. Moreover, A␤ disrupts axonal transport by a mechanism that it is initiated by NMDARs and mediated by GSK-3␤ [25]. In addition to interacting with NMDARs, A␤ peptides modulate and/or activate recombinant and native AMPA receptors [14,26], but the nature of those interactions remains unclear. The present study, however, indicates unambiguously that in the absence of glutamate, A␤ oligomers activate both NR1/NR2A and NR1/NR2B NMDARs.NMDA-type glutamate receptors mediate many forms of synaptic plasticity. These tetrameric receptors consist of two obligatory NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B subunits. In the neonatal neocortex, NR2B-containing NMDARs predominate, and sensory experience facilitates a developmental switch in which NR2A levels increase relative to NR2B [27]. Our findings reveal that the rel-


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Fig. 3. Currents activated by amyloid beta (A␤) oligomers are blocked by NMDA receptor antagonists. (A) A␤-activated currents in NR1/NR2A-expressing oocytes were blocked by incubating the oocytes with D-AP5, MK-801, or memantine. (B) Amplitude and electrical charge values when A␤ (1 ␮M) and glycine (10 ␮M) were co-applied to NR1/NR2A-injected oocytes incubated without (positive control, 100%) or with NMDA receptor antagonists. (C) A␤-activated currents in NR1/NR2B-expressing oocytes were also reduced by NMDA receptor antagonists. (D) Amplitude and electrical charge values when A␤ (1 ␮M) and glycine (10 ␮M) were co-applied to NR1/NR2B-injected oocytes incubated without (positive control, 100%) or with NMDA receptor antagonists *p ≤ 0.05, **p ≤ 0.01.

ative amplitude and current charge elicited by A␤ oligomers in oocytes expressing NR1/NR2A are higher than in oocytes expressing NR1/NR2B. This suggests preferential activation of NR1/NR2A receptors by A␤ oligomers. NR1/NR2A and NR1/NR2B receptors differ in their channel kinetics, synaptic localization, and protein binding partners, all of which influence synaptic plasticity. Thus, synapses that possess a high NR2A/NR2B ratio favor the induction of long-term depression versus long-term potentiation by limiting Ca2+ entry through NMDARs [28]. In a disease scenario in which A␤ oligomers accumulate, such as in AD, this may result in overall synaptic silencing, memory loss, cognitive decline and, ultimately, neuronal death. Indeed, there is significantly lower expression of

NR2A and NR2B in susceptible regions of the AD brain, whereas expression of NR2C and NR2D do not differ from controls [27]. However, there are no apparent differences in the reduction of the expression of NR2A and NR2B subunits. A␤ oligomers induce neurotoxicity in cortical and hippocampal neurons, and this toxicity is attenuated by NMDA receptor antagonists [14]. In the current study, we show that that native NMDA receptors activated by A␤ oligomers preferably lack NR2B subunits, a feature which may help to develop new antagonist with therapeutic potential. Several reports have described that antiglutamatergic treatment reduces clinical deterioration in moderate-to-severe AD [29,30]. In addition, a sustained increase in extracellular glutamate

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Fig. 4. A␤ oligomers induce Ca2+ influx into cortical neurons in culture by activating preferably NMDA receptors lacking NR2B subunit. (A) A␤ (5 ␮M) induces a rapid increase in [Ca2+ ]i , an effect that is blocked by NMDA receptor antagonist AP5 and slightly attenuated by ifenprodil, a blocker of NR2B-selective antagonist. (B) Histogram showing the quantitative effects of AP5 and ifenprodil on A␤ oligomer-induced increases in cytosolic Ca2+ levels. *p ≤ 0.05, ***p ≤ 0.001.

levels and activation of the NMDA receptor is associated with the cognitive deficits and loss of neurons observed in brains of AD patients [31,32]. The results presented here suggest that direct activation of NMDARs by A␤ oligomers may contribute to neuronal loss in AD. In addition to neurons, astrocytes and oligodendrocytes also express functional NMDARs [33–35], and glial cells are involved in the pathogenesis and progression of neurodegenerative diseases, including AD [36]. Thus, activation of NMDARs in glial cells by A␤ oligomers may also contribute to AD pathogenesis. In summary, we have provided evidence that A␤ oligomers in the absence of glutamate activate directly recombinant NMDARs expressed in Xenopus oocytes. These findings contribute to the notion that A␤ contributes to AD pathogenesis and that A␤ load correlates with the progression of tissue loss and cognitive decline observed in AD. The fact that NMDA receptors activated by A␤ oligomers preferably lack NR2B subunits may help to develop new selective antagonists with higher therapeutic efficacy. Acknowledgments This work was supported by CIBERNED and by grants from Ministerio de Educación y Ciencia (SAF2007/62380 and SAF2008/732), La Marató de TV3 063033, and Gobierno Vasco. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ceca.2011.02.001. References [1] L.M. Billings, S. Oddo, K.N. Green, J.L. McGaugh, F.M. LaFerla, Intraneuronal A␤ causes the onset of early Alzheimer’s disease-related cognitive deficits in transgenic mice, Neuron 45 (2005) 675–688. [2] S. Oddo, A. Caccamo, T. Tran, M.P. Lambert, C.G. Glabe, W.L. Klein, F.M. LaFerla, Temporal profile of amyloid-␤ (A␤) oligomerization in an in vivo model of Alzheimer disease: a link between A␤ and tau pathology, J. Biol. Chem. 281 (2006) 1599–1604. [3] Y.M. Kuo, M.R. Emmerling, C. Vigo-Pelfrey, T.C. Kasunic, J.B. Kirkpatrick, G.H. Murdoch, M.J. Ball, A.E. Roherk, Water-soluble A␤ (N-40, N-42) oligomers in normal and Alzheimer disease brains, J. Biol. Chem. 271 (1996) 4077–4081. [4] R. Kayed, E. Head, J.L. Thompson, T.M. McIntire, S.C. Milton, C.W. Cotman, C.G. Glabe, Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis, Science 300 (2003) 486–489. [5] Y. Gong, L. Chang, K.L. Viola, P.N. Lacor, M.P. Lambert, C.E. Finch, G.A. Krafft, W.L. Klein, Alzheimer’s disease-affected brain: presence of oligomeric A␤ ligands (ADDLs) suggests a molecular basis for reversible memory loss, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 10417–10422.

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