Non-specific Detection of a Major Western Blotting Band in Human Brain Homogenates by a Multitude of Amyloid Precursor Protein Antibodies.

Hazal Haytural,Mohammed Seed Ahmed,Jolanta L Lundgren,Bengt Winblad,Lars O Tjernberg,Tansu B Köse,Marinela Kalcheva,Erik Sundström,Gaël Barthet,Susanne Frykman,Tomàs Jordà-Siquier

doi:10.3389/fnagi.2019.00273

Abstract

The use of human post-mortem brain material is of great value when investigating which pathological mechanisms occur in human brain, and to avoid translational problems which have for example been evident when translating animal research into Alzheimer disease (AD) clinical trials. The amyloid β (Aβ)-peptide, its amyloid precursor protein (APP) and the intermediate APP-c-terminal fragments (APP-CTFs) are all important players in AD pathogenesis. In order to elucidate which APP CTF that are the most common in brain tissue of different species and developmental stages, and whether there are any differences in these fragments between AD and control brain, we investigated the occurrence of these fragments using different APP c-terminal antibodies. We noticed that whereas the conventional APP-CTFα and CTFβ fragments were most prominent in rat and mouse brain tissue, the major western blotting band detected in human, macaque and guinea pig was of approximately 20 kDa in size, possibly corresponding to the newly discovered APP-CTFη. However, this band was also intensely stained with a total protein stain, as well as by several other antibodies. The staining intensity of the 20 kDa band by the APP antibodies varied considerably between samples and correlated with the staining intensity of this band by the total protein stain. This could potentially be due to non-specific binding of the antibodies to another protein of this size. In-gel digestion and mass spectrometry confirmed that small amounts of APP were present in this band, but many other proteins were identified as well. The major hit of the mass spectrometry analysis was myelin basic protein (MBP) and a myelin removal protocol removed proportionally more of the 20 kDa APP band than the full-length APP and APP-CTFα/β bands. However, the signal could not be immunodepleted with an MBP antibody. In summary, we report on a potentially non-specific western blotting band of approximately 20 kDa and call for precaution when analyzing proteins of this size in human brain tissue.

Highlights

Alzheimer disease (AD) is the most common form of dementia and causes extensive suffering for millions of patients and their relatives
Brain tissues from frontal cortex Brodmann area 9 (BA9) of 10 sporadic AD and 10 control cases were obtained from Brains for Dementia Research, London, UK, while the human brain tissue used for the experiments with the different antibodies, immunoprecipitation and different species was obtained from the Brain Bank at Karolinska Institutet, Stockholm, Sweden
The 20 kDa band corresponds to the expected weight of the proposed amyloid precursor protein (APP)-CTFη, generated by η-secretase cleavage at N504 in the APP695 sequence (Willem et al, 2015)

Summary

Introduction

Alzheimer disease (AD) is the most common form of dementia and causes extensive suffering for millions of patients and their relatives. The only medication available gives just subtle symptomatic relief, and a large number of clinical trials of disease-modifying drugs have failed in the last decade. This is partially due to problems with translation, i.e., compounds that work well in mouse models fail in clinical trials. Non-amyloidogenic pathway, APP is cleaved by the α-secretase a disintegrin and metalloproteinase 10 (ADAM10) instead of BACE1 This cleavage produces the 83 amino acids long CTFα which can be cleaved by the γ-secretase complex, precluding Aβ production since the α-cleavage site is situated within the Aβ sequence (Postina et al, 2004)

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Conclusion