Objectives: To understand the cleavage of the amyloid b protein (Aβ) precursor (APP) by γ-secretase and to determine its changes in a representative familial Alzheimer disease (FAD) mutation. Methods: Transfected cells expressing wild-type and FAD mutant APP were analyzed for changes in the levels of the major secreted Aβ species and of the corresponding intracellular C-terminal APP fragments (APP intracellular domain, AICD) generated by γ-secretase, whereas radio-sequencing was used to precisely identify the resulting cleavage site(s). Results: The AICD fragment(s) generated by γ-secretase cleavage comigrated in gels with a 50-residue synthetic peptide used as control, which is smaller than the 59 and 57 residues predicted from Aβ ending at positions 40 (Aβ40) and 42 (Aβ42), respectively. In agreement with previous findings, an FAD mutant form of presenilin 1 (PS1-M139V) significantly increased the longer Aβ42 while showing trends toward reducing Aβ40. AICD levels were reduced by the mutation, suggesting that γ-secretase activity may be actually impaired by the mutation. Radiosequence analysis in cells expressing wild-type PS1 detected γ-secretase cleavage sites at the Ab peptide bond L49-V50 to generate a 50- amino acid (aa) AICD fragment (AICD50) and the Aβ peptide bond T48-L49, generating an AICD of 51 aa (AICD51). No other cleavage sites were reliably detected. Conclusions: Based on findings that the FAD mutation that increases Aβ42 also reduces AICD, we propose that γ-secretase activity is impaired by FAD mutations and predict that physiologic and environmental agents that inhibit γ-secretase will actually induce AD pathogenesis rather that prevent it. Furthermore, we propose that the cleavage site to generate AICD is naturally ragged and occurs predominantly at two sites 48 and 49 aa from the start of the Ab sequence. Thus, end specific antibodies to these two sites will need to be generated to study the quantitative relationships between these two cleavages in sporadic AD and FAD.
- Alzheimer disease
- Carboxy-terminal fragment (CTF)-γ
ASJC Scopus subject areas
- Psychiatry and Mental health
- Geriatrics and Gerontology