Cell Calcium 51 (2012) 95–106 Contents lists available at SciVerse ScienceDirect Cell Calcium j ourna l ho me page: www.elsevier.com/locate/ceca Amyloid beta peptide 1–42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures I.L. Ferreira a , L.M. Bajouco a , S.I. Mota a , Y.P. Auberson c , C.R. Oliveira a,b , A.C. Rego a,b,∗ a CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal b Faculty of Medicine, University of Coimbra, Coimbra, Portugal c Novartis Institutes of Biomedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland a r t i c l e i n f o Article history: Received 18 April 2011 Received in revised form 4 November 2011 Accepted 17 November 2011 Available online 15 December 2011 Keywords: Alzheimer’s disease Amyloid beta peptide (A) Calcium N-Methyl-d-aspartate receptor GluN2A subunit GluN2B subunit a b s t r a c t Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that leads to debilitating cognitive deficits. Recent evidence demonstrates that glutamate receptors are dysregulated by amyloid beta pep- tide (A) oligomers, resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits. Although it is well accepted that neuronal death in AD is related to disturbed intracel- lular Ca 2+ (Ca 2+ i ) homeostasis, little is known about the contribution of NMDARs containing GluN2A or GluN2B subunits on A-induced Ca 2+ i rise and neuronal dysfunction. Thus, the main goal of this work was to evaluate the role of NMDAR subunits in dysregulation of Ca 2+ i homeostasis induced by A 1–42 preparation containing both oligomers (in higher percentage) and monomers in rat cerebral cortical neurons. The involvement of NMDARs was evaluated by pharmacological inhibition with MK-801 or the selective GluN2A and GLUN2B subunit antagonists NVP-AAM077 and ifenprodil, respectively. We show that A, like NMDA, increase Ca 2+ i levels mainly through activation of NMDARs containing GluN2B subunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca 2+ i rise induced by a high concentra- tion of A (1 M), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca 2+ i homeostasis. Moreover, A modulate NMDA-induced responses and vice versa. Indeed, pre-exposure to A (1 M) decrease NMDA-evoked Ca 2+ I rise and pre-exposure to NMDA decrease A response. Inter- estingly, simultaneous addition of A and NMDA potentiate Ca 2+ I levels, this effect being regulated by GluN2A and GluN2B subunits in opposite manners. This study contributes to the understanding of the molecular basis of early AD pathogenesis, by exploring the role of GluN2A and GluN2B subunits in the mechanism of A toxicity in AD. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Alzheimer’s disease (AD) is the leading cause of dementia in western countries and the most prevalent neurodegenerative disease in the elderly population, affecting 26.6 million people worldwide [1]. Age-related forms of dementia lead to sporadic AD. Conversely, less than 10% of cases are associated with familial AD, due to mutations in either amyloid precursor protein, presenilin-1 or presenilin-2 genes. AD hallmarks include atrophy in the cortex, hippocampus and amygdala [2]. Neuropathologically, AD is char- acterized by senile plaques, composed of extracellular deposits of ∗ Corresponding author at: CNC-Center for Neuroscience and Cell Biology Univer- sity of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal. Tel.: +351 239 820190; fax: +351 239 822776. E-mail addresses: a.cristina.rego@gmail.com, arego@fmed.uc.pt, acrego@cnc.uc.pt (A.C. Rego). amyloid-beta peptides (A) and intracellular neurofibrillary tan- gles formed by hyperphosphorylated tau [2,3]. A is produced by proteolytic cleavage of APP by sequential activity of - and - secretases, producing A1–42 and A1–40 [4,5]. Neurodegeneration and synaptic dysfunction induced by A involves overactivation of the N-methyl-d-aspartate (NMDA) receptors (NMDARs) resulting in the elevation of intracellular Ca 2+ i levels, a process named excitotoxicity [6–10]. Activation of NMDARs was hypothesized to occur at late-stage AD, when plaque formation is expected. However, recent reports strongly suggest that glutamate receptors are dysregulated by A accumulation in the initial stages of AD, resulting in disruption of glutamater- gic synaptic transmission, which parallels early cognitive deficits [11]. Thus, early phases of AD (characterized by the presence of A monomers and oligomers) are linked to NMDAR-induced synaptic dysfunction, which appears to precede neurodegeneration [7,12–14]. Accordingly, recent studies suggest that A oligomers are the main neurotoxic species involved early in AD [15,16]. 0143-4160/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2011.11.008