The major emphasis of this project is the discovery of the role of EAA receptors in neurodegenerative disorders of aging. This year we identified additional isoforms of the NMDAR1 receptor subunit and we measured changes in receptor mRNA isoform expression by RNase protection. We detected an approximate 20% increase in hippocampus in an isoform lacking several phosphorylation sites in the carboxyl terminal in aged rats. Mini-gene constructs of the GLUR-2 glutamate/AMPA receptor subunit were constructed and transfected into cultured cells in order to examine the mutually exclusive splicing of two exons important in regulating agonist sensitivity of this subunit. We discovered that eliminating a downstream branch point led to exon exclusion and surprisingly, use of an upstream cryptic donor site. We cloned and characterized the promoter region of the NMDAR1 receptor. We identified two major transcription start sites at nucleotides -276 and -238 upstream of codon 1. Proximal to the transcriptional start site are one GSG and two SP1 regulatory motifs but no TATA or CAAT boxes. These results suggest that the NMDAR1 receptor gene has characteristics of a housekeeping gene but may be regulated by immediate-early genes. Reporter gene constructs of this promoter region show strong positive and strong negative elements. We are currently studying the role of NGF in regulating NMDAR1 gene expression.
