An antisense construct reduces N-methyl-D-aspartate receptor 2A expression and receptor-mediated excitotoxicity as determined by a novel flow cytometric approach.
The N-methyl-D-aspartate receptor (NMDAR) is a major neurotransmitter receptor in the central nervous system (CNS), with functional roles in learning, memory, and sensation. Several mechanisms potentiate NMDARs, and NMDAR hyperexcitability plays pathophysiological roles in many conditions, such as neurodegenerative disease, ischemia, and chronic conditions arising from spinal cord injury. Previous research suggests that the NR2A subunit of the receptor contributes to NMDAR excitotoxicity in heterologous cells and in neurons in vivo. To investigate the role of NR2A in NMDAR excitotoxicity, we have developed a system based on flow cytometry that allows rapid evaluation of the effect of antisense constructs on protein expression and channel function. The enhanced yellow fluorescent protein (EYFP) was fused to obligatory NMDAR subunits, allowing expression to be monitored in living cultured cells. An NR2A antisense construct, asNR2A, specifically and effectively reduced NR2A-EYFP expression. NR1 and NR2A fusion proteins formed functional, excitotoxic channels upon co-expression. The asNR2A RNA significantly reduced NMDAR excitotoxicity when NR2A levels were limiting for channel formation. Using our assay system, further optimization can be achieved rapidly. The asNR2A construct and the assays developed for this study can be used to provide insights into NMDAR biology and disease.
Mattar
Pierre A
PA
BioTherapeutics Research Group, The John P. Robarts Research Institute, London, Ontario, Canada.
Holmes
Kevin D
KD
Dekaban
Gregory A
GA
D000818:Animals; D001426:Bacterial Proteins; D002460:Cell Line; D016373:DNA, Antisense; D004622:Embryo, Mammalian; D005434:Flow Cytometry / Q000379:methods*; D022781:Hepatocytes / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D001426:Bacterial Proteins; D016373:DNA, Antisense; D008164:Luminescent Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria