Analysis of transcriptional regulatory sequences of the N-methyl-D-aspartate receptor 2A subunit gene in cultured cortical neurons and transgenic mice

Abstract

The postnatal appearance and up-regulation of the NR2A subunit of the N-methyl-D-aspartate receptor contributes to the functional heterogeneity of the receptor during development. To elucidate the molecular mechanisms that regulate the neural and developmental specific expression of NR2A, an upstream ∼9-kb region of the gene harboring the promoter was isolated and characterized in transgenic mice and transfected cortical neurons. Transgenic mouse lines generated with luciferase reporter constructs driven by either 9 or 1 kb of upstream sequence selectively transcribe the transgene in brain, as compared with other non-neural tissues. Reporter luciferase levels in dissociated cultures made from these mice are over 100-fold greater in neuronal/ glial co-cultures than in pure glial cultures. Analysis of NR2A 5′-nested deletions in transfected cultures of cortical neurons and glia indicate that while sequences residing upstream of - 1079 bp augment NR2A neuronal expression, sequences between -486 and -447 bp are sufficient to maintain neuronal preference. An RE1/ NRSE element is not necessary for NR2A neuron specificity. Furthermore, comparison of the 5′-deletion constructs in cortical neurons grown for 5, 8, 11, or 14 days in vitro indicate that sequences between -1253 and -1180 bp are necessary for maturational up-regulation of NR2A. Thus, different cis-acting sequences control the regional and temporal expression of NR2A, implicating distinct regulatory pathways.