Presenilin-directed inhibitors of c-secretase trigger caspase 3 activation in presenilin-expressing and presenilin-deficient cells Cristine Alves da Costa, * Erwan Ayral, Jean-Franc ¸ois Hernandez, Peter St George-Hyslopà and Fre ´de ´ric Checler * * Institut de Pharmacologie Mole ´culaire et Cellulaire of Centre National de la Recherche Scientifique, Valbonne, France  LAPP, CNRS UMR5810, 15, Avenue Charles Flahault, Montpellier, France àTanz Neurosciences Building, Toronto, Canada Abstract The amyloid b peptide (Ab) is generated by subsequent cleavages by b- and c-secretases. Therefore, these two enzymes are putative therapeutic targets to prevent Ab pro- duction, and hopefully to slow down or even stop the Alzhei- mer’s disease (AD) neurodegenerative process. Several studies have revealed that c-secretase hydrolyses other important substrates besides b-amyloid precursor protein (bAPP) thus adding another level of complexity to designing fully AD-specific interfering drugs. Here we demonstrate that three distinct presenilin-directed c-secretase inhibitors as well as JLK compounds indirectly potentiate caspase 3 activity, the effector caspase of the apoptotic cascade. Thus, inhibitors were shown to drastically stimulate caspase 3 activity in wild- type mice blastocyst-derived and fibroblast cells. Interestingly, some of these inhibitors known to interact with presenilins also trigger caspase activation in presenilin-deficient cells. How- ever, inhibitors do not affect recombinant caspase 3 activity, indicating that the effect on this enzyme was indirect. Fur- thermore, we established that caspase 3 activation was not due to an effect of c-secretase inhibitors on calpains, a family of proteolytic enzymes able to modulate caspase 3 activity. Altogether, our data demonstrate that presenilin-directed c-secretase inhibitors affect caspase 3 activity in a presenilin- independent manner. Therefore, as presenilin-dependent c-secretase activity is not specific for bAPP and because its inhibitors clearly affect other vital cell functions, care should be taken in considering ‘c-secretase’ inhibitors as putative therapeutic tools to interfere with AD pathology. Keywords: caspase 3, c-secretase, inhibitors, presenilins, presenilins-knockout. J. Neurochem. (2004) 90, 800–806. The exact aetiology of Alzheimer’s disease (AD) is still doubtful but transgenic approaches, mutational analyses and cell biology studies have suggested that the overpro- duction of the amyloid b-peptide (Ab) if not causative, appears to be at least closely associated with this pathology ( Selkoe 2001; Suh and Checler 2002). Ab is a natural catabolite of bAPP that is liberated from its precursor by the sequential attacks by b- and c-secretases two generic terms referring to one or a group of enzymes generating the N- and C-termini, respectively (Checler 1995; Octave 1995; Hardy and Selkoe 2002). The nature of this C-terminal moiety varies and conditions Ab toxicity and aggregation properties. However the 42-amino acid long Ab counterpart is clearly the most pathogenic species. These observations underline the importance of the two secretase enzymes as putative therapeutic targets. The b-secretase referred to as BACE (b-site APP-cleaving enzyme, ASP2 or memapsin 2) has been identified as a novel aspartyl protease (for reviews see (Citron 2001; Vassar 2001; John et al. 2003). The nature of the c- secretase is still a matter for discussion (Checler 2001; Sisodia et al. 2001; Wolfe 2001a; Sisodia and St George- Hyslop 2002) but clearly, part of the activity is borne by a multiproteic complex including at least presenilins (Taka- sugi et al. 2003), nicastrin (Yu et al. 2000), and two recently described proteins, APH-1 and Pen 2 (Francis Received November 28, 2003; revised manuscript received March 16, 2004; accepted March 17, 2004. Address correspondence and reprint requests to Fre ´de ´ric Checler, Institut de Pharmacologie Mole ´culaire et Cellulaire, UMR6097 dl CNRS, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. E-mail: checler@ipmc.cnrs.fr Abbreviations used:Ab, amyloid b-peptide; AD, Alzheimer’s disease; BACE, b-site APP cleaving enzyme; BAPP, b amyloid precursor pro- tein. Journal of Neurochemistry , 2004, 90, 800–806 doi:10.1111/j.1471-4159.2004.02512.x 800 Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 800–806