Down-regulation amyloid beta-protein 42 production by interfering with transcript of presenilin 1 gene with siRNA.
OBJECTIVE
To investigate the pathogenesis of Abeta42 yielding and new drug targets as well as the possibility of RNA interference (RNAi) technique for treatment of Alzheimer disease (AD).


METHODS
Human AD presenilin 1 (PS1) cDNA sequence was obtained from NCBI website. The three sites of RNAi action and one missense control site were selected in PS1 cDNA through online design of Ambion company. To confirm specificity of these sites, we conducted a BLAST search of the IMAGE EST library. The corresponding double-stranded DNA was used to construct pSilencer 3.1-H1 plasmid, which could transcribe small interference RNA (siRNA). Then, the pSliencer 3.1-H1 plasmids were transfected into CHO/PS1/APP cells with SuperFect transfection reagent. The cells have been transfected with the mutant PS1 and APP gene of AD. All the CHO/PS1/APP cells with pSliencer 3.1-H1 plasmids were screened out using G418. Transcripts of PS1 gene in CHO/PS1/APP were measured by RT-PCR, the contents of PS1 peptide and Abeta42 production inside CHO/PS1/APP cells were examined through Western blot and the Abeta42 change of secretion by CHO/PS1/APP was determined with ELISA.


RESULTS
The site 3 of PS1 mRNA was inhibited by RNAi after 2 d. The effect was more obvious with the time. The peptide corresponding to PS1 gene and Abeta42 production in CHO/PS1/APP cells were both reduced after siRNA interfere for 3 d. Abeta42 secretion by CHO/PS1/APP cells began to reduce on d 3, and reached the most significance on d 5. There was a time-dependent relationship between the transcript of PS1 gene and the production of Abeta42 with RNAi action.


CONCLUSIONS
PS1 is essential for g-secretase activity. Inhibition of the PS1 can decrease the levels of Abeta42. Some sites of PS1 mRNA, for example, the site 3, may serve as a new drug target and RNAi probably can be used for treatment of AD.
Luo
Huan-min
HM
Neuropharmacological Research Laboratory, College of Pharmacy, Ji-nan University, Guangzhou 510632, China. tlhm@jnu.edu.cn
Deng
Hui
H
Xiao
Fei
F
Gao
Qin
Q
Weng
Wen
W
Zhang
Pei-fen
PF
Li
Xiao-guang
XG
D000544:Alzheimer Disease / Q000235:genetics; D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D016466:CHO Cells / Q000201:enzymology / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism*; D053764:Presenilin-1; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014158:Transcription, Genetic; D014162:Transfection
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D010446:Peptide Fragments; D053764:Presenilin-1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C075222:amyloid beta-protein (1-42); D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human