The Effects of Antisense Presenilin-l and BACE Expression on the Production of 𝛽-Amyloid in Transfected Cells
Wells, Deonna G.
The cerebral deposition of 𝛽-amyloid (A𝛽) peptide is a characteristic feature of Alzheimer's disease (AD). A𝛽 formation requires the subsequent proteolytic cleavage of the 𝛽-amyloid precursor protein (APP) by both 𝛽- and 𝛾-secretases. Although several groups have identified the main 𝛽-secretase as the 𝛽-site APP cleaving enzyme (BACE), the identity of the 𝛾-secretase( s) is still controversial .. Recent studies have implicated both BACE and PS1 in APP processing, and it has been suggested that a presenilin-containing complex is responsible for 𝛾-secretase activity. To address the interactions between APP processing and these proteases, we constructed two different human cell lines, one expressing a constitutive antisense PS1 RNA and the other, a constitutive antisense BACE RNA .. The effect of each cell line on normal levels of PS1 or BACE and AP was determined via Western Blots and ELISA. In this study a statistically significant unexpected elevation in both A𝛽40 and A𝛽42 for both cell lines was observed. For both the antisense BACE and PS1 expressing lines, no statistically significant changes were detected in the respective protein levels. From these results it is unclear if the antisense construct was working successfully. In order to make this determination, farther analysis is warranted. A polymerase chain reaction (PCR) based Taqman analysis of both cell lines should be used to determine if the antisense RNAs are actually being made. Repetition of the experiments using stable independent cell lines may also provide useful information to ongoing research addressing the interaction between PS1 and BACE and APP processing.
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