Evidence Of A Novel Mechanism For Partial Gamma-Secretase Inhibition Induced Paradoxical Increase In Secreted Amyloid Beta Protein

BACE1 (beta-secretase) and alpha-secretase cleave the Alzheimer's amyloid beta protein (A beta) precursor (APP) to C-terminal fragments of 99 aa (CTF beta) and 83 aa (CTF alpha), respectively, which are further cleaved by gamma-secretase to eventually secrete A beta and A alpha (a.k.a. P3) that terminate predominantly at residues 40 and 42. A number of gamma-secretase inhibitors (GSIs), such as N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), have been developed with the goal of reducing A beta to treat Alzheimer's disease (AD). Although most studies show that DAPT inhibits A beta in a dose-dependent manner several studies have also detected a biphasic effect with an unexpected increase at low doses of DAPT in cell cultures, animal models and clinical trials. In this article, we confirm the increase in A beta 40 and A beta 42 in SH-SY5Y human neuroblastoma cells treated with low doses of DAPT and identify one of the mechanisms for this paradox. We studied the pathway by first demonstrating that stimulation of A beta, a product of gamma-secretase, was accompanied by a parallel increase of its substrate CTF beta, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an A beta degrading activity, endothelin converting enzyme (ECE), yielded more A beta, but Abolished the DAPT-induced stimulation. Finally, we have demonstrated that A alpha, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of gamma-secretase can paradoxically increase A beta by transiently skirting A beta degradation in the endosome. This study adds to the growing body of literature suggesting that preserving gamma-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration.