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.