TAILIEUCHUNG - Báo cáo khoa học: Selecting cells with different Alzheimer’s disease c-secretase activity using FACS Differential effect of presenilin exon 9 deletion on c- and e-cleavage

The ultimate step in Alzheimer’s disease Abgeneration involves c-secretase, which releases Abfrom its membrane-bound precursor. Asimilar presenilin-dependent proteolytic activity is implicated in the release of theNotch intracellular domain. We have developed a novel assay forc-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. | Eur. J. Biochem. 270 495-506 2003 FEBS 2003 doi Selecting cells with different Alzheimer s disease c-secretase activity using FACS Differential effect of presenilin exon 9 deletion on c- and e-cleavage M. Fleur Sernee1 Genevieve Evin1. Janetta G. Culvenor1. Jose A Villadanaos2 Konrad Bevreuther3. B BB Colin Lb Masters1 and Roberto Cappai1 1 Department of Pathology The University of Melbourne and The Mental Health Research Institute Parkville Victoria Australia 2The Walter and Eliza Hall Institute of Medical Research Parkville Victoria Australia 3Center for Molecular Biology ZMBH University of Heidelberg Heidelberg Germany The ultimate step in Alzheimer s disease Ab generation involves y-secretase which releases Ab from its membranebound precursor. A similar presenilin-dependent proteolytic activity is implicated in the release of the Notch intracellular domain. We have developed a novel assay for y-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 y-secretase activity with high fluorescent cells producing more Ab than low fluorescent cells. Specific y-secretase inhibitors L-685 458 and MW167 reduced cell fluorescence in a dose-dependent manner that paralleled inhibition of Ab secretion. Overexpression of presenilin 1 increased the cell fluorescence. Cells expressing presenilin with different aspartate mutations D257A D385A and D257A D385A or exon 9 deletion mutation showed reduced fluorescence. The single aspartate mutations showed a concomitant reduction in Ab secretion whereas the D257A D385A and AE9 mutations had no effect on Ab secretion. Keywords secretase amyloid .

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