Generation of C‐terminally truncated amyloid‐β peptides is dependent on γ‐secretase activity

Abstract

Aberrant production of amyloid-b peptides by processing of the b-amyloid precursor protein leads to the formation of characteristic extracellular protein deposits which are thought to be the cause of Alzheimer's disease. Therefore, inhibiting the key enzymes responsible for amyloid-b peptide generation, b-and c-secretase may offer an opportunity to intervene with the progression of the disease. In human brain and cell culture systems a heterogeneous population of amyloid-b peptides with various truncations is detected and at present, it is unclear how they are produced. We have used a combination of surface enhanced laser desorption/ionization timeof-flight mass spectrometry (SELDI-TOF MS) and a specific inhibitor of c-secretase to investigate whether the production of all amyloid-b peptide species requires the action of c-secretase. Using this approach, we demonstrate that the production of all truncated amyloid-b peptides except those released by the action of the nonamyloidogenic a-secretase enzyme or potentially beta-site bAPP cleaving enzyme 2 depends on c-secretase activity. This indicates that none of these peptides are generated by a separate enzyme entity and a specific inhibitor of the c-secretase enzyme should have the potential to block the generation of all amyloidogenic peptides. Furthermore in the presence of c-secretase inhibitors, the observation of increased cleavage of the membrane-bound bAPP C-terminal fragment C99 by a-secretase suggests that during its trafficking C99 encounters compartments in which a-secretase activity resides. Keywords: Alzheimer's disease, bAPP processing, c-secretase inhibitor, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Amyloid-b (Ab) peptide is the major proteinaceous constituent of extracellular protein deposits which are characteristic of Alzheimer's disease (AD). These deposits occur either as senile plaques in the brain parenchyma or as vascular amyloid around the brain blood vessels (Glenner and Wong 1984;Masters et al. 1985). Ab peptides themselves are generated by processing of the b-amyloid precursor protein (bAPP) (Kang et al. 1987) which requires consecutive cleavages involving b-and c-secretase enzymes (Haass et al. 1992). An alternative processing pathway leads to the release of the bAPP ectodomain as secretory bAPP by cleavage within the Ab domain thereby excluding Ab peptide formation (Weidemann et al. 1989). Members of the disintegrin and metalloprotease family (ADAM) such as ADAM10 and TACE appear to mediate this alternative cleavage termed a-secretase cleavage Lammich et al. 1999). The recently cloned b-secretase enzyme BACE1 (Asp-2) (Hussain et al. 1999;Sinha et al. 1999;Vassar et al. 1999;Yan et al. 1999;Lin et al. 2000) has been shown to generate the membrane bound bAPP C-terminal fragment (C99) intermediate, which is a prerequisite for the release of Ab peptide by c-secretase as the final processing step. BACE1 is the major b-secretase responsible for the generation of Ab peptides by neurones (Cai et al....

Longer Forms of Amyloid β Protein: Implications for the Mechanism of Intramembrane Cleavage by γ-Secretase

“…Various A␤ species, including A␤37, A␤38, A␤39, A␤40, and A␤42, were present in the conditioned media, an observation consistent with previous reports (Wang et al, 1996;Clarke et al, 1998;Beher et al, 2002) (Fig. 2 A).…”

Random Mutagenesis of Presenilin-1 Identifies Novel Mutants Exclusively Generating Long Amyloid β-Peptides

“…The results in Fig. 6A confirm previous reports (30,35,36) and show that, in MEFs expressing WT-PS1, the peptidomimetic difluoroketone inhibitor DFK167 selectively enhanced the ␥-secretase activity of A␤ 42/43 generation (referred to as ␥-42/43 activity) at subinhibitory doses, but inhibited the ␥-secretase activity of A␤ 40 generation (referred to as ␥-40 activity) in a dose-dependent manner. However, this enhancement of A␤ 42/43 secretion was , n ϭ 3)).…”

“…This is the case for PS1 LA mutants, which are distinctive in mediating exclusively ␥-43 cleavage of APP, but not self-endoproteolysis, ␥-40 cleavage, and ⑀-cleavage. A␤ 43 is a minor A␤ product of normal ␥-secretase activity, but the longest identified so far, and its secretion, like that of A␤ 42 , is associated with pathogenic PS1 mutations (33) or DFK167 treatment (36).…”

Differential Effects between γ-Secretase Inhibitors and Modulators on Cognitive Function in Amyloid Precursor Protein-Transgenic and Nontransgenic Mice

“…This attenuation may be due to a shift from ␤-cleavage to ␣-cleavage of APP, as the concurrent increase in sAPP␣ and decrease in sAPP␤ in the media were seen by ELISA (data not shown). Alternatively, enhanced cleavage of ␤-CTF by ␣-secretase could also be a possible explanation (Beher et al, 2002;Cook et al, 2010;Portelius et al, 2011).…”