Results

As a prototype γ‐secretase inhibitor, we investigated the highly specific γ‐secretase inhibitor DAPT (Dovey et al., 2001) for its capacity to block Notch endoproteolysis. With this aim, HEK293 cells expressing endogenous PSs were stably transfected with the NotchΔE cDNA [encoding a tagged version of the transmembrane and intracellular domains of Notch (Schroeter et al., 1998)] and treated with or without DAPT. As shown in Figure 1A, the NICD fragment is readily visible in untreated cells, but its generation is inhibited by DAPT treatment. Next, before testing the effects of DAPT in the zebrafish in vivo, we verified that DAPT was also active on a zebrafish PS1 (zfPS1)‐controlled γ‐secretase activity. We stably transfected HEK293 cells expressing Swedish mutant APP (HEK293/sw)(Citron et al., 1992) with cDNA encoding wild‐type (wt) zfPS1 or the non‐functional zfPS1 D374N mutant (Leimer et al., 1999). As expected (Leimer et al., 1999), zfPS1 was endoproteolytically processed, while endoproteolysis of zfPS1 D374N was blocked and the full‐length protein accumulated (Figure 1B). Endogenous human PS1 and PS2 were replaced (Thinakaran et al., 1997) by wt and mutant zfPS1, demonstrating that zfPS1 is incorporated into the PS complex, the formation of which is required for γ‐secretase activity (Li et al., 2000a)(Figure 1B). Expression of zfPS1 D374N caused a dramatic accumulation of the substrates of γ‐secretase, the APP C‐terminal fragments (CTFs), which was accompanied by an almost complete inhibition of total Aβ (Aβ40 and Aβ42) generation (Figure 1B). In contrast, expression of wt zfPS1 did not cause APP CTF accumulation and allowed normal total Aβ production (Figure 1B). We next analyzed Notch endoproteolysis in HEK293/sw cells stably co‐expressing NotchΔE and wt or D374N mutant zfPS1. Expression of wt zfPS1 allowed robust NICD production, which was strongly inhibited by the zfPS1 D374N mutant (Figure 1C). Taken together, these results demonstrate that zfPS1 is capable of controlling γ‐secretase and S3 protease activity in human cells. We therefore next investigated the effects of DAPT on APP and Notch endoproteolysis in the presence of zfPS1‐controlled γ‐secretase and S3 protease activity. DAPT caused a strong accumulation of APP CTFs with concomitant inhibition of Aβ generation (Figure 1D). DAPT also inhibited S3 cleavage of Notch in the presence of zfPS1. As shown in Figure 1E, NICD production was inhibited by DAPT in a dose‐dependent manner. Thus, these experiments demonstrate that DAPT efficiently blocks a zfPS1‐dependent γ‐secretase and S3 protease activity.