Analysis of the 5 '-flanking region of the beta-amyloid precursor protein gene that contributes to increased promoter activity in differentiated neuronal cells

To study the transcription control of the beta-amyloid precursor protein (b eta APP) in Alzheimer's disease (AD), we functionally characterized the bet a APP gene promoter in differentiated cells. PC12 cells were first differen tiated with nerve growth factor (NGF) and then transient transfection analy sis was done with a series of 5'-deletion constructs, that extended as far upstream as -7900 down to + 104 base pair (bp) relative to the transcriptio n start site (+1). The truncated regions of the promoter were linked upstre am to a reporter gene, chloramphenicol acetyl transferase (CAT). The CAT as say was performed to compare promoter activity of different 5'-fanking and intronic regions of the beta APP gene. Our results suggest that the longest (-7900/+104) and one of the shortest (-47/+104) regions possessed signific antly higher levels of promoter activity than the promoterless vector in NG F-differentiated PC12 cells. A deletion of about 7600 bp region from the -7 900 to +104 construct resulted in 50% loss of original promoter activity. A deletion of all but 47 bp from the -7900 to +104 construct resulted in the loss of 66% (and retention of 34%) promoter activity. The region -3416/+10 4 bp displayed the strongest promoter activity whereas +1/+104 bp showed th e least activity among all deletion constructs studied. The upstream region -5529 to -3416 contains a negative regulatory element and -3416 to -1131 c ontains a positive regulatory element. The very upstream region, -7900 to - 3411, lacks independent functional activity. The 5'-UTR region (+1 to +104) showed minimum activity and the -75 to +104 region constitutes the basic p romoter element. The first exon or a large part of the first intron (+99 to +6200) did not display any significant promoter activity. Thus, several po sitive and negative regulatory elements influence the basal level of beta A PP promoter activity in NGF-differentiated PC12 cells. We speculate that an y structural alteration(s) due to a specific mutation in these regulatory r egions can potentially alter the transcriptional machinery, and that can pe rhaps affect the level of beta-amyloid protein involved in AD. (C) 2000 Els evier Science B.V. All rights reserved.