Major Carboxyl Terminal Fragments Generated by γ-Secretase Processing of the Alzheimer Amy loid Precursor Are 50 and 51 Amino Acids Long

Objectives: To understand the cleavage of the amyloid beta protein (A beta) precursor (APP) by gamma-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 A beta species and of the corresponding intracellular C-terminal APP fragments (APP intracellular domain, AICD) generated by gamma-secretase, whereas radio-sequencing was used to precisely identify the resulting cleavage site(s). Results: The AICD fragment(s) generated by gamma-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 A beta ending at positions 40 (A beta 40) and 42 (A beta 42), respectively. In agreement with previous findings, an FAD mutant form of presenilin 1 (PS1-M139V) significantly increased the longer A beta 42 while showing trends toward reducing A beta 40. AICD levels were reduced by the mutation, suggesting that gamma-secretase activity may be actually impaired by the mutation. Radiosequence analysis in cells expressing wild-type PS1 detected gamma-secretase cleavage sites at the A beta peptide bond L-49-V-50 to generate a 50-amino acid (aa) AICD fragment (AICD50) and the A beta peptide bond T-48-L-49, generating an AICD of 51 aa (AICD51). No other cleavage sites were reliably detected. Conclusions: Based on findings that the FAD mutation that increases A beta 42 also reduces AICD, we propose that gamma-secretase activity is impaired by FAD mutations and predict that physiologic and environmental agents that inhibit gamma-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 A beta 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. (Am J Geriatr Psychiatry 2013; 21:474-483)