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

Abstract

Pathogenic generation of the 42-amino acid variant of the amyloid ␤-peptide (A␤) by ␤-and ␥-secretase cleavage of the ␤-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of A␤ 42 production by ␥-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the A␤ domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of ␥-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the A␤ domain regarding their response to GSMs. We found that A␤ 42 -increasing familial AD mutations of the ␥-secretase cleavage site domain responded robustly to A␤ 42 -lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of A␤ 42 produced. We thus expect that familial AD patients carrying mutations at the ␥-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine-scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as other A␤ species (A␤ 41 , A␤ 39 ) could also be lowered besides A␤ 42 . Moreover, certain mutations simultaneously increased A␤ 42 and the shorter peptide A␤ 38 , arguing that the proposed precursor-product relationship of these A␤ species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in A␤ 42 generation (Gly-29, Gly-33) and potentially in GSM-binding (Lys-28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.Alzheimer disease (AD) 3 is the most common neurodegenerative disorder worldwide. The ␤-amyloid precursor protein (APP), a type I membrane protein, plays a central role in the pathogenesis of the disease (1). Sequential cleavage of APP by ␤-and ␥-secretase generates the amyloid-␤ (A␤) peptide, which deposits as plaques in the brain of affected patients and represents one of the principal pathological hallmarks of the disease (1). ␥-Secretase is an intramembrane-cleaving protease complex, which cleaves the APP transmembrane domain (TMD) in a progressive, stepwise manner via cleavages at the ⑀-, -, and ␥-sites until it is sufficiently shortened to allow the release of A␤ from the membrane (2-4). A␤ peptides generated by ␥-secretase cleavage differ in their C termini. The major product released is A␤ 40 , whereas A␤ 38 and A␤ 42 represent minor species (1). The highly aggregation-prone, neurotoxic A␤ 42 is believed to be causative for AD by initiating a cascade of pathogenic events, which ultimately causes neurodegeneration and dementia (1). Increased production of A␤ 42 underlies the vast majority of mutations associated with familial AD (FAD), which manifests with a very early disease onset. The majority of FAD mutations have been found in PS1, the catalytic subunit of ␥-secretase (5), whereas only a few mutations were found in its h...

Attenuated Aβ42 Responses to Low Potency γ-Secretase Modulators Can Be Overcome for Many Pathogenic Presenilin Mutants by Second-generation Compounds

“…Point mutations disrupting key residues within the proposed GSM binding site in APP did not abolish the efficacy of GSMs (13). Likewise, both familial and synthetic mutations at the ␥-secretase cleavage site domain showed the typical GSM responses (13). In contrast, although FAD mutations in PS still increase A␤ 38 in response to NSAIDs, lowering of A␤ 42 is not effective for the majority of these mutants (9,22,23).…”

“…Secreted A␤ species were analyzed by sandwich immunoassay specific for A␤ 38 , A␤ 40 , and A␤ 42 species (Meso Scale Discovery), Tris-Bicine urea SDS-PAGE, and MALDI-TOF mass spectrometry as described previously (13). IC 50 values of GSMs for inhibition of A␤ 42 were determined from dose-response curves by nonlinear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.…”

“…In addition to A␤ 42 , the longer A␤ 43 (e.g. by flurbiprofen) and also the shorter A␤ 39 and A␤ 40 as well as the rare A␤ 41 species can be decreased by GSMs, suggesting a complex mechanism of GSM action (13). Of note, inverse modulators have also been identified that increase the longer A␤ 42 species while lowering the shorter A␤ 38 species (14).…”

“…Mutational analyses of APP do not seem to support substrate binding of GSMs either (13). Point mutations disrupting key residues within the proposed GSM binding site in APP did not abolish the efficacy of GSMs (13). Likewise, both familial and synthetic mutations at the ␥-secretase cleavage site domain showed the typical GSM responses (13).…”

“…However, controversial data challenging the view of substrate-targeting GSMs were also reported (10,17) and supported a number of studies that had initially suggested that GSMs target the enzyme (12, 18 -21). Mutational analyses of APP do not seem to support substrate binding of GSMs either (13). Point mutations disrupting key residues within the proposed GSM binding site in APP did not abolish the efficacy of GSMs (13).…”