Selecting cells with different XXXX gamma-secretase activity using FACS. Differential effect on presenilin exon 9 gamma- and epsilon-cleavage.

@entity46372 @entity72213 @entity3666 @entity195 @entity1658 @entity45672
The ultimate step in @entity195 Abeta generation involves gamma-secretase, which releases Abeta from its membrane-bound precursor. A similar presenilin-dependent proteolytic activity is implicated in the release of the Notch intracellular domain. We have developed a novel assay for gamma-secretase activity based on green fluorescent protein detection. This involves cotransfection of a substrate-activator based on the amyloid precursor protein or the Notch sequence and a fluorescent reporter gene. Stable fluorescent cell populations were selected by fluorescent activated cell sorting and characterized. This assay enabled the identification and sorting of populations, which differ in their levels of gamma-secretase activity, with high fluorescent cells producing more Abeta than low fluorescent cells. Specific gamma-secretase inhibitors, @entity1658 and @entity45672 , reduced cell fluorescence in a dose-dependent manner that paralleled inhibition of Abeta secretion. Overexpression of @entity3666 increased the cell fluorescence. Cells expressing presenilin with different aspartate mutations ( @entity46372 , @entity72213 and @entity46372 / @entity72213 ) or exon 9 deletion mutation showed reduced fluorescence. The single aspartate mutations showed a concomitant reduction in Abeta secretion, whereas the @entity46372 / @entity72213 and DeltaE9 mutations had no effect on Abeta secretion.
