Amyloid beta peptide 1–42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures

Abstract

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 peptide (Aβ) 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 intracellular Ca²⁺ (Ca²⁺_i) homeostasis, little is known about the contribution of NMDARs containing GluN2A or GluN2B subunits on Aβ-induced Ca²⁺_i rise and neuronal dysfunction. Thus, the main goal of this work was to evaluate the role of NMDAR subunits in dysregulation of Ca²⁺_i homeostasis induced by Aβ 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 Aβ, like NMDA, increase Ca²⁺_i levels mainly through activation of NMDARs containing GluN2B subunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca²⁺_i rise induced by a high concentration of Aβ (1 μM), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca²⁺_i homeostasis. Moreover, Aβ modulate NMDA-induced responses and vice versa. Indeed, pre-exposure to Aβ (1 μM) decrease NMDA-evoked Ca²⁺_I rise and pre-exposure to NMDA decrease Aβ response. Interestingly, simultaneous addition of Aβ and NMDA potentiate Ca²⁺_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 Aβ toxicity in AD.

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 characterized by senile plaques, composed of extracellular deposits of amyloid-beta peptides (Aβ) and intracellular neurofibrillary tangles formed by hyperphosphorylated tau [2], [3]. Aβ is produced by proteolytic cleavage of APP by sequential activity of β- and γ-secretases, producing Aβ1–42 and Aβ1–40 [4], [5].

Neurodegeneration and synaptic dysfunction induced by Aβ involves overactivation of the N-methyl-d-aspartate (NMDA) receptors (NMDARs) resulting in the elevation of intracellular Ca²⁺_i levels, a process named excitotoxicity [6], [7], [8], [9], [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 Aβ accumulation in the initial stages of AD, resulting in disruption of glutamatergic synaptic transmission, which parallels early cognitive deficits [11]. Thus, early phases of AD (characterized by the presence of Aβ monomers and oligomers) are linked to NMDAR-induced synaptic dysfunction, which appears to precede neurodegeneration [7], [12], [13], [14]. Accordingly, recent studies suggest that Aβ oligomers are the main neurotoxic species involved early in AD [15], [16]. Indeed, oligomeric species were shown to be more toxic to cortical neurons than fibrillar forms, the main components of senile plaques [17]. In addition, it has been reported that Aβ1–42 oligomers are much more prone to aggregation and are more neurotoxic than those composed by the Aβ1–40 peptide [5], [18].

NMDAR functional downregulation is thought to take place during the initial stages of AD. Supporting this view, many studies have suggested that Aβ may reduce surface GluN1 subunit of NMDARs, impairing its function [19], [20], [21], leading to the depressed synaptic glutamatergic transmission observed in AD.

Three families of genes (GluN1, GluN2 and GluN3) have been identified that encode NMDAR subunits [22]. Functional NMDARs are heterotetramers composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 (GluN2A-GluN2D) subunits, or in some cases GluN3 (GluN3A and/or GluN3B) subunits, the latter replacing the GluN2 subunits [23]. The most widely expressed NMDARs contain the obligatory subunit GluN1 plus either GluN2B or GluN2A or a mixture of the two. GluN2B and GluN2D are expressed at high levels in early developmental stages (prenatally), whereas GluN2A and GluN2C expression is first detected near birth [24]. In adults, GluN2A is ubiquitously expressed in the brain, GluN2B is mostly restricted to the forebrain, GluN2C is restricted to the cerebellum, and GluN2D is expressed in small numbers of cells in selected brain regions [25].

It has been recently proposed that GluN2A- and GluN2B-containing NMDAR are linked to different intracellular cascades, participating in different functions, from synaptic plasticity to pathological conditions [26], [27], [28]. Although it is well accepted that neuronal death in AD is related to disturbed Ca²⁺_i homeostasis involving the NMDAR [6], [10], [29], [30] and that early NMDAR dysregulation occur in AD, little is known about the contribution of the GluN2A or GluN2B subunits on Aβ-induced neuronal dysfunction. In this work we evaluated the contribution of the NMDARs subunits GluN2A and GluN2B, on Ca²⁺_i dysregulation induced by direct exposure to Aβ1–42. This study supports the hypothesis that GluN2A and/or GluN2B subunits of the NMDARs are important in the initial stages of AD pathogenesis and are involved in neuronal dysfunction induced by Aβ, leading to cortical neurodegeneration.