Toxicity of β-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl- d-aspartate receptor subunits

• Published in: Neurochemistry international Vol. 50; no. 6; pp. 872 - 880
• Main Authors: Domingues, A., Almeida, S., da Cruz e Silva, E.F., Oliveira, C.R., Rego, A.C.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.05.2007; Elsevier
• Subjects: Adenosine Triphosphate - metabolism; Alzheimer's disease; Amyloid beta-Peptides - toxicity; Biological and medical sciences; Blotting, Western; Calcium - metabolism; Calcium influx; Caspases - metabolism; Cell death; Cell Line; Cell Survival - physiology; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Dizocilpine Maleate - pharmacology; DNA Fragmentation; Excitatory Amino Acid Antagonists - pharmacology; Excitotoxicity; Fundamental and applied biological sciences. Psychology; Humans; Immunohistochemistry; L-Lactate Dehydrogenase - metabolism; Medical sciences; Neurology; NMDA receptors; Receptors, N-Methyl-D-Aspartate - genetics; Receptors, N-Methyl-D-Aspartate - metabolism; Stimulation, Chemical; Transfection; Vertebrates: nervous system and sense organs; NMDA receptors; Cell death; Alzheimer's disease; Excitotoxicity; Calcium influx; Nervous system diseases; Calcium; Alzheimer disease; Toxicity; Glutamate receptor; Subunit; Cerebral disorder; β Amyloid protein; Central nervous system disease; Degenerative disease; NMDA receptor
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/j.neuint.2007.03.001

Neurotoxicity induced by beta-amyloid peptide (Aβ) involves glutamate toxicity, resulting from overactivation of N-methyl- d-aspartate (NMDA) receptors and elevation of intracellular calcium. However, the heterogeneity of the NMDA receptors, frequently composed of NR1 and NR2A-D subunits, has been less studied. Thus, we determined the contribution of NMDA receptor subtypes on Aβ 1–40 toxicity in HEK293 cells transiently expressing NR1/NR2A or NR1/NR2B subunits. Analysis of lactate dehydrogenase (LDH) release and trypan blue exclusion revealed an increase in Aβ 1–40 toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Aβ 1–40 decreased intracellular ATP in cells expressing NR1/NR2A. MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[ a, d]cyclohepten-5,10-imine maleate), a noncompetitive NMDA receptor antagonist, partially prevented the decrease in cell viability and the energy impairment. These differences were not accounted for by the activation of caspases 2, 3, 8 and 9 or calpains or by DNA fragmentation, excluding the hypothesis of apoptosis. Functional NR1/NR2A and NR1/NR2B receptor subtypes were further evidenced by single-cell calcium imaging. Stimulation of NR1/NR2A receptors with NMDA/glycine revealed an increase in intracellular calcium in cells pre-exposed to Aβ 1–40. Opposite effects were observed upon activation of NR1/NR2B receptors. These results suggest that NR1/NR2A-composed NMDA receptors mediate necrotic cell death in HEK293 cells exposed to Aβ 1–40 through changes in calcium homeostasis.