Amyloid β peptide oligomers directly activate NMDA receptors
Introduction
Soluble oligomers of the amyloid-β (Aβ) peptide are neurotoxins that contribute to Alzheimer's disease (AD) pathology. Aβ 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], [5], [6]. The abundance of Aβ oligomers also correlates with disease progression [7], [8], [9]. Aβ 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]. Aβ 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, Aβ oligomer neurotoxicity is neutralized by NMDA receptor antagonists, both in dissociated neurons and in organotypic hippocampal cultures [14].
Since Aβ peptides can regulate the release of glutamate [15], the deleterious effects of Aβ oligomers could be caused by over-activation of NMDA receptors due to excessive glutamate at synapses. However, silencing neuronal activity with tetrodotoxin and removal of extracellular Ca2+ to minimize transmitter release does not have any effect on the NMDA receptor-mediated currents induced by Aβ oligomers [14]. Because of that observation, we hypothesized that Aβ 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 Aβ 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.
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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).
Expression of NMDA receptors comprised of NR1/NR2A and NR1/NR2B subunits
The plasmids containing the cDNA for human NR1 (pCI-neo), NR2A (HumNR2A-pcDNAI/Amp) and NR2B (HumNR2B6-pcDNAI/Amp) subunits were
Effects of Aβ oligomers on oocytes injected with water (control)
We first studied the interaction of soluble Aβ 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 μM) plus glycine (10 μM) or glycine (10 μM) alone was added. No currents were observed in the water-injected oocytes (Fig. 1A). Similarly, no currents were observed when Aβ oligomers (1 μM) were added with glycine (Fig. 1A).
Aβ oligomers elicit inward currents in NR1/NR2A-injected oocytes
We next
Discussion
The results presented here show that in the absence of glutamate, Aβ 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 Aβ 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
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.
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