In a separate, non-amyloidogenic pathway, APP is cleaved by the α-secretase a disintegrin and metalloproteinase 10 (ADAM10) instead of BACE1. The CTFβ is further cleaved by the protein complex γ-secretase, generating Aβ and the APP intracellular domain (AICD Selkoe, 2011). After the first cleavage by β-site APP cleaving enzyme 1 (BACE1), the main β-secretase of neurons, the 99 or 89 amino acid long c-terminal fragment (CTFβ) remains in the membrane while the soluble APPβ (sAPPβ) is released. Aβ is generated by the cleavage of the 695–770 amino acid long amyloid precursor protein (APP) by first β-secretase and then the γ-secretase complex. The synaptotoxic amyloid β-peptide (Aβ) plays a critical role in the pathophysiology of AD ( Selkoe, 2011 Prince et al., 2013). Thus, it is of utmost importance to study the alterations in human AD brain. This is partially due to problems with translation, i.e., compounds that work well in mouse models fail in clinical trials. Unfortunately, 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. 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.Īlzheimer disease (AD) is the most common form of dementia and causes extensive suffering for millions of patients and their relatives. However, the signal could not be immunodepleted with an MBP antibody. 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. 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. This could potentially be due to non-specific binding of the antibodies to another protein of this size. 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. However, this band was also intensely stained with a total protein stain, as well as by several other 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. 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. 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. 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. 3Wolfson Centre for Age-Related Diseases, King’s College London, London, United Kingdom.2Interdisciplinary Institute of Neuroscience, Université de Bordeaux, Bordeaux, France.1Division of Neurogeriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Science and Society, Karolinska Institutet, Solna, Sweden.Köse 1, Tomàs Jordà-Siquier 2, Marinela Kalcheva 1, Mohammed Seed Ahmed 1,3‡, Bengt Winblad 1, Erik Sundström 1, Gaël Barthet 2, Lars O.
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