Affiliations: [a] Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden | [b] Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, the Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden | [c] UCL Institute of Neurology, Queen Square, London, UK
Correspondence:
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Correspondence to: Erik Portelius, PhD, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, S-431 80 Mölndal, Sweden. Tel.: +46 31 3430025; Fax: +46 31 3432426; E-mail: [email protected].
Note: [1] These authors contributed equally to this work.
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.
Keywords: Alzheimer's disease, amyloid-β, biomarkers, dimers, mass spectrometry, oligomers
