Independent Generation of Aβ42 and Aβ38 Peptide Species by γ-Secretase

Abstract

Proteolytic processing of the amyloid precursor protein by ␤-and ␥-secretase generates the amyloid-␤ (A␤) peptides, which are principal drug targets in Alzheimer disease therapeutics. ␥-Secretase has imprecise cleavage specificity and generates the most abundant A␤40 and A␤42 species together with longer and shorter peptides such as A␤38. Several mechanisms could explain the production of multiple A␤ peptides by ␥-secretase, including sequential processing of longer into shorter A␤ peptides. A novel class of ␥-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower A␤42 levels without a change in A␤40 levels. A signature of GSMs is the concomitant increase in shorter A␤ peptides, such as A␤38, leading to the suggestion that generation of A␤42 and A␤38 peptide species by ␥-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished A␤42 reductions as compared with wild type PS1. We have now used this model system to investigate whether A␤38 production would be similarly affected indicating coupled generation of A␤42 and A␤38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased A␤38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other A␤ peptide species. These data provide evidence that A␤42 and A␤38 species can be independently generated by ␥-secretase and argue against a precursor-product relationship between these peptides.A variety of therapeutic strategies in clinical development for Alzheimer disease (AD), 2 the most common neurodegenerative disorder, target the amyloid-␤ (A␤) peptides that are generated through proteolytic processing of the transmembrane amyloid precursor protein (APP) (1). In the A␤-producing pathway, APP is cleaved by two aspartyl proteases, first by ␤-secretase within its ectodomain and subsequently by ␥-secretase, which cleaves APP within its transmembrane domain (TMD) (2). ␥-Secretase is a multiprotein complex with the presenilin (PS) proteins at its enzymatic core (2). Because of its imprecise cleavage specificity, ␥-secretase generates A␤ peptides of variable length at the carboxyl terminus, with the highly amyloidogenic A␤42 isoform thought to be the key pathogenic species (3). A central role of A␤42 developed largely from genetic research demonstrating that mutations in the APP and PS genes associated with early-onset familial AD (FAD) invariably increase the A␤42/A␤40 ratio in primary fibroblast...

β-Amyloid Precursor Protein Mutants Respond to γ-Secretase Modulators

“…Based on our data, we therefore conclude that treatment with GSMs might provide a successful therapeutic option also for these mutant carriers and others of the ␥-secretase cleavage site domain with pathogenic A␤ 42 production that may be identified in the future. Interestingly, our results contrast with those obtained recently for PS mutations (24,25). Most of the PS FAD mutations investigated were not responsive to NSAIDs that act as A␤ 42 -lowering GSMs, such as sulindac sulfide.…”

“…Mechanistically, NSAIDs that lower A␤ 42 were suggested to allosterically alter the conformation of PS (6,10,44), and conformational changes of PS opposite to that induced by such NSAIDs were also observed for PS FAD mutants (6,7,10). It is thus conceivable that many aggressive PS FAD mutations are locked in a conformation that makes the PS-substrate interaction refractory to the A␤ 42 -lowering capacity of GSMs (24,25). Clearly, as shown in this study, mutations in the APP substrate are permissive to GSMs, suggesting that the substrate is conformationally more flexible than the ␥-secretase enzyme, allowing APP substrate positioning such that the ␥42 site is less exposed to the active site of ␥-secretase.…”

“…In particular, aggressive FAD mutants that manifest with a very early disease onset due to their strongly increased A␤ 42 production do not respond at all, whereas increased A␤ 38 production was nevertheless still observed (24,25). Whether or not APP FAD mutants respond to GSMs has not been conclusively studied yet, however (27).…”

“…However, a recent study demonstrated that dimerization per se might not be a factor that determines ␥-secretase cleavage specificity (22), and whether GXXXG mutants inhibit dimerization is controversial (23). In addition, the lack of a consistent response to GSMs regarding an inversely correlated production of A␤ 42 and A␤ 38 by PS FAD mutants observed earlier argued against a strict precursor-product relationship between A␤ 42 and A␤ 38 (24,25). Moreover, while this manuscript was in preparation, a biophysical study failed to demonstrate GSMbinding to the APP substrate, however, and suggested that the reported GSM-APP interaction (18) was unspecific (26).…”

Alzheimer Presenilin-1 Mutations Dramatically Reduce Trimming of Long Amyloid β-Peptides (Aβ) by γ-Secretase to Increase 42-to-40-Residue Aβ

“…The expected tri-and tetrapeptide products have been detected by mass spectrometry (18). Despite the evidence, however, other studies suggest that A␤ and AICD production can be dissociated (20,21) or that there is no precursorproduct relationship between A␤42 and A␤38 (22,23). The former contention has been disproven by the determination of equimolar A␤ and AICD production (24), and the latter has been unambiguously settled recently with the demonstration that synthetic A␤42 is converted to A␤38 by isolated ␥-secretase (25).…”