Dominance of Amyloid Precursor Protein Sequence over Host Cell Secretases in Determining β-Amyloid Profiles Studies of Interspecies Variation and Drug Action by Internally Standardized Immunoprecipitation/Mass Spectrometry

Abstract

␤-Amyloid peptides, tentatively regarded as the principal neurotoxins responsible for Alzheimer's Disease, make up a set of products that varies significantly among different biological systems. The full implications of this complexity and its variations have yet to be defined. In this work, A␤ peptide populations were extracted from animal brain tissue or cell-conditioned media, immunoprecipitated with specific antibodies, and analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. 15N-Substituted A␤ internal standards were added to gauge variations in the profile of captured peptides. Results from a range of species, including guinea pig, dog, rabbit, and wild-type and transgenic mice, showed that the A␤ peptide population in each system was mainly determined by the species of origin of the amyloid precursor protein (APP) and not by the host tissue or cell line. The same method was used to gauge the effect on the A␤ peptide profile of an inhibitor of ␥-secretase, one of the two proteinases that excises A␤ peptides from the precursor protein with different effects on specific peptides. Overall, the results demonstrate that the species of origin of the APP substrate dictates the outcome of APP processing to a greater extent than the origin of the processing enzymes, an important consideration in rationalizing the properties of different model systems.A␤ peptides are tentatively regarded as the principal neurotoxins responsible for AD (Watson et al., 2005) and are formed by sequential proteolytic events near the C terminus of the 770-residue APP. They are also the main components of amyloid plaques that are the classical biomarker of AD in post mortem brain. In the first step of pathological processing, the membrane-bound aspartic proteinase BACE cleaves APP after Met-671 to generate a C-terminal fragment called APP-C-terminal fragment ␤. A␤ species are liberated from this fragment by the intramembrane proteinase ␥-secretase. Both secretases are seen as important drug targets, because the amyloid hypothesis of AD implies that blocking the production of A␤ peptides will retard the disease (Gandy, 2005). The alternative ␣-secretory pathway does not lead to A␤ peptide production.

Pharmacodynamics and Pharmacokinetics of the γ-Secretase Inhibitor PF-3084014

“…At doses that reduce A␤1-40 and A␤1-42, DAPT [N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester] increases the production of A␤1-43, A␤1-46, and other longer A␤ fragments in vitro (Qi-Takahara et al, 2005;Yagishita et al, 2006). PF-3084014 similarly exhibited an elevation of A␤1-43 at doses that reduced levels of multiple other A␤ peptides, a finding previously reported in guinea pig brain after LY-411575 treatment (Du et al, 2007). DAPT, LY-411575, and LY-450139 all have been shown to preferentially inhibit A␤1-40 over A␤1-42 and elevate A␤ levels at low concentrations (Lanz et al, 2003(Lanz et al, , 2004Burton et al, 2008).…”

“…A␤ was immunoprecipitated from Tg2576 and guinea pig brain extracts and analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy as described previously (Du et al, 2007).…”

“…The Tg2576 model expresses APP with the human Swedish mutation, although the rate of overexpression may not be identical to that of the cell line used in the whole-cell assay. In addition to the production of more A␤, the panoply of A␤ peptides and A␤40/42 ratios produced in Tg2576 mouse differ from those of guinea pig (Du et al, 2007). Thus confirmation of both safety and efficacy endpoints in the same species is vital to establishing a therapeutic window.…”

Selective modulation of amyloid‐β peptide degradation by flurbiprofen, fenofibrate, and related compounds regulates Aβ levels

“…Immunoprecipitation-MALDI-TOF methods have been reported for Ab (Kametani et al 1999;Du et al 2007 (Du et al 2007): 4G8 binds the 17-24 epitope of human and murine amyloid peptides, whereas 6E10 recognizes Ab 4-8 of human amyloid peptides. Sample extraction and work-up conditions were optimized for optimal S/N in the mass spectra of untreated cell supernatants.…”

ACAT1 gene ablation increases 24(S)-hydroxycholesterol content in the brain and ameliorates amyloid pathology in mice with AD

“…These results led us to conclude that A1− leads to reduction in only the hAPP level, not the mAPP level. Subtle sequence differences exist between hAPP and mAPP, and these differences may play important roles in causing differential fates of hAPP and mAPP (30,31). We investigated the effect of A1− on mutant human tau (htau) in 3XTg-AD mice.…”