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

Abstract

Objectives To understand the cleavage of the amyloid β protein (Aβ) precursor by γ-secretase and determine its changes in a representative Familial (FAD) Alzheimer’s disease (AD) 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 radiosequencing was used to precisely identify the resulting cleavage site(s). Results The AICD fragment(s) generated by γ-secretase cleavage co-migrated in gels with a 50 residue synthetic peptide used as control being, therefore, smaller than the expected 59 and 57 residues based on the classical Aβ size 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 towards 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 Aβ peptide bond L49-V50 to generate a 50 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 physiological and environmental agents that inhibit γ-secretase will actually induce AD pathogenesis rather that preventing 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 Aβ 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.

APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression

“…The γ-secretase activity in living cells was calculated by subtracting the unspecific turnover determined by adding 2 μM of the highly specific γ-secretase inhibitor X (Shearman et al, 2000 ). It should be mentioned that similar results showing a decreased γ-secretase activity for several PS1 FAD mutations were observed by us and others in previous studies confirming our assay and the obtained data (Walker et al, 2005 ; Wiley et al, 2005 ; Bentahir et al, 2006 ; Pinnix et al, 2013 ; Grimm et al, 2014 ).…”

“…In contrast to the APPswe mutation affecting β-secretase cleavage, mutations in the catalytically active presenilins affect γ-secretase processing of APP, mainly by shifting the Aβ42/40 ratio towards the generation of the more hydrophobic Aβ42 species (Duff et al, 1996 ; Citron et al, 1997 ; Duering et al, 2005 ). However, several studies have revealed that some of the known PS mutations also reduce γ-secretase processing of APP resulting in decreased AICD levels compared to cells expressing wt presenilins (Walker et al, 2005 ; Wiley et al, 2005 ; Bentahir et al, 2006 ; Pinnix et al, 2013 ). As a genetic approach we analyzed PS-deficient MEFs retransfected with the FAD PS1 mutation T354I (MEF PS1-T354I) compared to PS1 wt (MEF PS1rescue).…”

“…Processing of α-CTF and β-CTF fragments by the γ-secretase complex also results in the release of the APP intracellular domain (AICD) to the cytosol which is discussed to be involved in gene transcription (Passer et al, 2000 ; Gao and Pimplikar, 2001 ; Cao and Sudhof, 2004 ; von Rotz et al, 2004 ; Pardossi-Piquard and Checler, 2012 ). Multiple-site cleavages executed by the γ-secretase complex generate accordingly AICDs of various lengths including C50, C53, C57, and C59 (Gu et al, 2001 ; Pinnix et al, 2001 ; Sastre et al, 2001 ; Yu et al, 2001 ; Sato et al, 2003 ; Zhang et al, 2012 ; Pinnix et al, 2013 ). However, all of the endogenous AICD isoforms are rarely detected, because of their rapid degradation in the cytosol by insulin degrading enzyme (IDE) (Edbauer et al, 2002 ; Farris et al, 2003 ; Miller et al, 2003 ), the proteasome (Nunan et al, 2003 ) and Cathepsin B (Vingtdeux et al, 2007 ; Asai et al, 2011 ).…”

γ-Secretase Processing and Effects of γ-Secretase Inhibitors and Modulators on Long Aβ Peptides in Cells

“…We would note that previous studies on M139V have led to different conclusions; a cell-free assay had shown that M139V does not alter ⑀-cleavage efficiency but does impair the fourth stepwise cleavage along both product lines when C99 was used as an in vitro substrate (36). In a separate report using a cell model, M139V increased A␤42 but also reduced ⑀-cleavage efficiency (54). However, our present results are consistent with a previous study from our group where we found that the M139V mutant selectively raised A␤42 but did not alter levels of other peptides in CHO cells stably overexpressing this mutant (55).…”

Arc Regulates Transcription of Genes for Plasticity, Excitability and Alzheimer’s Disease

“…13B). Arc's activation of the γ-secretase PSEN1 to promote cleavage of APP not only increases amyloid beta load, but also results in an increased level of the APP intracellular domain (AICD) 80,176 . AICD forms a complex with TIP60/Kat5 to alter transcriptional activity crucial for AD progression 150,[177][178][179][180][181][182] (Fig.…”