Potential Link between Amyloid β-Protein 42 and C-terminal Fragment γ 49–99 of β-Amyloid Precursor Protein

Abstract

A novel cleavage of ␤-amyloid precursor protein (APP), referred to as ⑀-cleavage, occurs downstream of the ␥-cleavage and generates predominantly a C-terminal fragment (CTF␥) that begins at Val-50, according to amyloid ␤-protein (A␤) numbering. Whether this cleavage occurs independently of, or is coordinated with, ␥-cleavage is unknown. Using a cell-free system, we show here that, although A␤40 and CTF␥ 50 -99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTF␥ 49 -99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of A␤42. Furthermore, a ␥-secretase inhibitor, which suppresses A␤40 production and paradoxically enhances A␤42 production at low concentrations, caused the proportion of CTF␥ 50 -99 to decrease and that of CTF␥ 49 -99 to increase significantly. These results strongly suggest a link between the production of A␤42 and CTF␥ 49 -99 and provide an important insight into the mechanisms of altered ␥-cleavage caused by mtAPP and mtPS1/2.Senile plaques, one of the neuropathological hallmarks of Alzheimer's disease (AD), 1 are composed primarily of amyloid ␤-protein (A␤) (1). Two major A␤ species consisting of 40 and 42 residues are generated mainly in neurons and constitutively secreted. A shorter one, A␤40, is predominant, and a longer one, A␤42, is a minor species (Ͻ10%) among secreted A␤ species. A␤ is produced from ␤-amyloid precursor protein (APP), through sequential cleavage by proteases referred to as ␤-and ␥-secretases. ␤-Secretase was identified as a type I membrane aspartic protease ␤-site APP-cleaving enzyme (BACE) (2), but the identity of ␥-secretase has remained unknown. ␥-Secretase cleaves APP in the middle of the transmembrane domain, releasing A␤ and its counterpart, C-terminal fragment ␥ of APP (CTF␥). Most recent studies have shown that ␥-secretase forms a large complex composed of presenilin (PS) 1 or 2, nicastrin, PEN-2, and APH-1, and the activity of ␥-secretase is now known to depend on these proteins (3-7).One of the A␤ species, A␤42, has a much higher aggregation potential (8, 9) and is believed to be initially deposited in senile plaques (10). It is reasonable to postulate that A␤42 accumulation in the brain is the very initial event in the development of AD including sporadic AD. Indeed, all mutations of PS1/2 and some mutations of APP that cause familial AD result in increased A␤42 production (11).Recently, we and other groups found that APP is cleaved by PS-dependent ␥-secretase, not only in the middle of the transmembrane domain (␥-cleavage) but also near the cytoplasmic membrane boundary (⑀-cleavage) (12-15). The major product of the latter process is a CTF␥ of APP that begins at Val-50. This cleavage site is a few residues inside the membrane from the cytoplasmic/membrane boundary and is similar to site 3 cleavage of Notch (16). Since production of CTF␥ is inhibited by a dominan...

Alzheimer’s disease mutations in APP but not γ-secretase modulators affect epsilon-cleavage-dependent AICD production

“…These results might be explained with the sequential cleavage model of APP-CTF, in which the generation of Ab40 is dependent on the AICD50-99, while Ab42/38 is linked to AICD49-99 (ref. 15). Based on this model, reduced AICD50-99 (as observed for some FAD mutants) is expected to be associated with less Ab40 and more Ab42/38, thus increasing the pathogenic ratio of Ab42/40.…”

“…The observed vertical shift in mutants (B2.5 Å) is in fact associated with a tilting of half of a helix turn (that is, displacement of two amino acids). Interestingly, Ab38 and Ab42 have been proposed to originate from the same sequential processing pathway, starting at the e-position 48, and directly linked to AICD49-99 production 15 . Consistent with this sequential model, we propose that the conformational changes in the T714I and V715A substrates, associated with reduced AICD50-99 levels, explain the preferential Ab38 and Ab42 production observed with these mutants.…”

“…1). After this step, gsecretase sequentially continues to trim the residual transmembrane stub, finally producing Ab42, Ab40 and Ab38 peptides 15 (Fig. 1).…”

APP mouse models for Alzheimer's disease preclinical studies

“…There is no evidence for an interaction between the Swedish mutation and Beyreuther/Iberian mutations, but the Arctic mutation increases CTFs (CTF‐β + CTF‐α) by 50% by unknown mechanisms (T Saito & TC Saido, unpublished; Cheng et al , 2004) and results in an unnatural Aβ conformation. It is important to perform delipidation pretreatment for the analysis of CTFs (Sato et al , 2003; Saito et al , 2014). …”

Coordinated Metabolism of Alcadein and Amyloid β-Protein Precursor Regulates FE65-dependent Gene Transactivation

“…The APP CTFs are secondarily cleaved at ␥-and/or ⑀-sites by the ␥-secretase complex, which results in the secretion of p3 or A␤ and releases the AICD (18). It is now well documented that the APP CTFs accumulate in the cell in the presence of the ␥-secretase inhibitor L-685,458 (Fig.…”