Toxicity of beta-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits.
Neurotoxicity induced by beta-amyloid peptide (Abeta) 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 Abeta(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 Abeta(1-40) toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Abeta(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 Abeta(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 Abeta(1-40) through changes in calcium homeostasis.
Domingues
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Institute of Biochemistry, Faculty of Medicine and Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal.
Almeida
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da Cruz e Silva
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Oliveira
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Rego
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D000255:Adenosine Triphosphate / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000633:toxicity*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D053938:DNA Fragmentation; D016291:Dizocilpine Maleate / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D013268:Stimulation, Chemical; D014162:Transfection
D016229:Amyloid beta-Peptides; D018691:Excitatory Amino Acid Antagonists; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D016291:Dizocilpine Maleate; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D020169:Caspases; D002118:Calcium