Mass Spectrometric Characterization of Amyloid-β Species in the 7PA2 Cell Model of Alzheimer's Disease

Abstract

The Chinese hamster ovary cell line 7PA2, stably transfected with the 751 amino acid isoform of amyloid-β protein precursor (AβPP) containing the Val → Phe mutation at residue 717, is one of the most used models to study the biochemistry and toxicity of secreted amyloid-β (Aβ) peptides, particularly Aβ oligomers, which are considered to be of relevance to the pathogenesis of Alzheimer's disease. Here, we present a detailed immunochemical and mass spectrometric characterization of primary structures of Aβ peptides secreted by 7PA2 cells. Immunoprecipitation and western blot of 7PA2 cell culture media revealed abundant anti-Aβ immunoreactive bands in the molecular weight range of 4-20 kDa. Mass spectrometric analysis showed that these bands contain several AβPP/Aβ peptides, starting at the N-terminal of the Aβ sequence and extending across the BACE1 cleavage site. Treatment of cells with a BACE1 inhibitor decreased the abundance of the Aβ monomer band by western blot and resulted in lower levels of Aβ1-40, Aβ1-42, and sAβPPβ as measured by ELISA. However, western blot bands thought to represent oligomers of Aβ increased in response to BACE1 inhibition. This increase was paralleled by the emergence of N-terminally truncated Aβ species (Aβ5-40 in particular) and Aβ species that spanned the β-secretase site in AβPP according to mass spectrometric analyses. The formation of these AβPP/Aβ peptides may have implications for the use of the 7PA2 cell line as a model for Aβ pathology. The enzyme(s) responsible for this particular BACE1-independent AβPP-processing remains to be identified.

Behavioral abnormalities in APPSwe/PS1dE9 mouse model of AD-like pathology: comparative analysis across multiple behavioral domains

“…However, in the light of recently published evidence suggesting that mice with significant amyloid burden but lacking APP processing derivatives could perform cognitive tasks with little or no deficits Melnikova et al, 2013), we are hesitant to link aggregated deposits of Ab as a cause of reduced burrowing activity. Nevertheless, our findings are consistent with the idea that Ab deposits might present a surrogate for the overall pathological changes related to amyloid aggregation and deposition, including soluble fractions of Ab and/or APP fragments, that mediate decline in mnemonic and non-mnemonic behaviors in transgenic APP rodent models (Mitani et al, 2012;Portelius et al, 2013;Tamayev and D'Adamio, 2012).…”

Retention in Endoplasmic Reticulum 1 (RER1) Modulates Amyloid-β (Aβ) Production by Altering Trafficking of γ-Secretase and Amyloid Precursor Protein (APP)

“…However, a protein fragment with a higher molecular mass (M r ϳ 8,000) than the A␤ monomer was detected (Fig. 5C), suggesting that A␤ oligomers or longer forms of A␤ peptide might be produced due to RER1 co-expression (58).…”

Truncated and modified amyloid-beta species

“…Interestingly, using APP-overexpressing cell lines, inhibition of BACE1 resulted in the appearance of Aβ5–40 [38,39]. This species has also been detected in the 5XFAD mouse model of AD [29] and in the CSF of dogs treated with BACE1 inhibitor [39].…”