title: Evidence of a novel mechanism for partial γ-secretase inhibition induced paradoxical increase in secreted amyloid β protein.
abstract: BACE1 (β-secretase) and α-secretase cleave the Alzheimer's amyloid β protein (Aβ) precursor (APP) to C-terminal fragments of 99 aa (CTFβ) and 83 aa (CTFα), respectively, which are further cleaved by γ-secretase to eventually secrete Aβ and Aα (a.k.a. P3) that terminate predominantly at residues 40 and 42. A number of γ-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 Aβ to treat Alzheimer's disease (AD). Although most studies show that DAPT inhibits Aβ 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 Aβ40 and Aβ42 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 Aβ, a product of γ-secretase, was accompanied by a parallel increase of its substrate CTFβ, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an Aβ degrading activity, endothelin converting enzyme (ECE), yielded more Aβ, but abolished the DAPT-induced stimulation. Finally, we have demonstrated that Aα, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of γ-secretase can paradoxically increase Aβ by transiently skirting Aβ degradation in the endosome. This study adds to the growing body of literature suggesting that preserving γ-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration.
authors.affiliation: Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Eliza
authors.initials: E
authors.lastname: Barnwell
authors.affiliation: Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Vasudevaraju
authors.initials: V
authors.lastname: Padmaraju
authors.affiliation: Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Baranello
authors.affiliation: Biomedical Research Institute of New Jersey, MidAtlantic Neonatology Associates and Atlantic Health System, Morristown, New Jersey, United States of America.
authors.forename: Javier
authors.initials: J
authors.lastname: Pacheco-Quinto
authors.affiliation: Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Craig
authors.initials: C
authors.lastname: Crosson
authors.affiliation: Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Ablonczy
authors.affiliation: Biomedical Research Institute of New Jersey, MidAtlantic Neonatology Associates and Atlantic Health System, Morristown, New Jersey, United States of America.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Eckman
authors.affiliation: Biomedical Research Institute of New Jersey, MidAtlantic Neonatology Associates and Atlantic Health System, Morristown, New Jersey, United States of America.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Eckman
authors.affiliation: Department of Biostatistics, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Ramakrishnan
authors.affiliation: Drug Design & Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, Baltimore, Maryland, United States of America.
authors.forename: Nigel H
authors.initials: NH
authors.lastname: Greig
authors.affiliation: Department of Neurology, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Pappolla
authors.affiliation: Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Kumar
authors.initials: K
authors.lastname: Sambamurti
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D011992:Endosomes / Q000502:physiology; D000072396:Endothelin-Converting Enzymes; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008954:Models, Biological; D059748:Proteolysis
chemical_list: D016229:Amyloid beta-Peptides; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes