Gene Expression Profiling in Cells with Enhanced ?-Secretase Activity

Magold, Alexandra I.; Cacquevel, Matthias; Fraering, Patrick C.

In an effort to identify specific alterations of gene transcription as a result of c-secretase activity, the transcriptomes of two CHO cell lines (biological triplicates were used in each case) with To identify genes whose transcription is affected by c-secretase activity, two starkly contrasting conditions were analyzed by cDNA microarray: genetically engineered enhanced c-secretase (left panel) and pharmacologically inhibited c-secretase (right panel) in CHO cell lines. For a schematic depiction of the strategy, the Notch-1 receptor signaling pathway is used as an example. After processing by the Furin protease and when activated by binding to its ligands Notch-1 is cleaved at the S2 position by the TACE protease, generating a substrate for c-secretase (1, 7) . Under enhanced (left panel) or inhibited (right panel) c-secretase activity, the cleavage of the substrate controls the release of the Notch intracellular domain (NICD) (2, 8) . With enhanced c-secretase, increased numbers of NICDs enter the nucleus and interact with CSL (3), leading to the transcription of target genes like Hes1 and Hey (4). The Hes1 transcription repressor inhibits transcription of target genes like NC3C1 (5), with the final consequence being reduced production of NC3C1 mRNA (6) . Thus, enhancing c-secretase leads simultaneously to gene-dependent increase (in the case of Hes/Hey) or decrease (in the case of NC3C1) of mRNA copy numbers. With inhibited c-secretase, reduced numbers of NICDs (9) lead to the transcription of less Hes1/Hey (10), to reduced inhibition of target genes like NC3C1 (11) and consequently to increased production of NC3C1 mRNA (12) . Inhibiting c-secretase thus leads to gene-dependent decrease (in the case of Hes/Hey genes) or increase (in the case of NC3C1) of mRNA copy numbers. Following mouse cDNA microarray analysis of both transcriptomes, top scoring candidates were evaluated and validated by real time PCR and further analyzed for changes of transcript levels between healthy and AD human brain cortices. doi:10.1371/journal.pone.0006952.g001 enhanced and inhibited c-secretase activity were analyzed and compared (strategy depicted in Fig. 1 -exemplified by Notch processing). The S-1 cell line overexpresses the four components of c-secretase (NCT, Aph1a, PS1 and Pen2) and was characterized by a marked increase in the level of PS1 heterodimers and an associated 8-fold increase in c-secretase activity compared to untransfected controls [17] . The other cell line consisted of the original parental wild type CHO cells incubated with DAPT, a well-known c-secretase inhibitor. We strategically chose those two conditions, overexpression of c-secretase and inhibition of its activity, to amplify the activity-dependent effects on gene transcription levels (i.e., amplification of the signal from the cDNA microarray). To reduce potential effects due to changes in the protein levels of the c-secretase subunits as opposed to changes in its activity that we are interested in, we used chemical inhibition (DAPT) of c-secretase activity instead of gene silencing, which ultimately leads to changes in protein levels [18] . Biological functions have indeed been reported mainly for the c-secretase subunit PS1, independently to the c-secretase activity. However, because treatment of CHO cells with DAPT has been recently reported to exacerbate the secretion of exosomes [19] , we cannot exclude at this stage that some detected genes may be exosomerelated in respo