beta-Amyloid Precursor Protein Mutants Respond to gamma-Secretase Modulators
ABSTRACT Pathogenic generation of the 42-amino acid variant of the amyloid beta-peptide (Abeta) by beta- and gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of Abeta(42) production by gamma-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Abeta domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of gamma-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the Abeta domain regarding their response to GSMs. We found that Abeta(42)-increasing familial AD mutations of the gamma-secretase cleavage site domain responded robustly to Abeta(42)-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Abeta(42) produced. We thus expect that familial AD patients carrying mutations at the gamma-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 Abeta species (Abeta(41), Abeta(39)) could also be lowered besides Abeta(42). Moreover, certain mutations simultaneously increased Abeta(42) and the shorter peptide Abeta(38), arguing that the proposed precursor-product relationship of these Abeta species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Abeta(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.
- SourceAvailable from: Taisuke Tomita
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- "A subset of NSAID-type GSMs have been reported to directly target the TMD of APP, especially the GXXXG motif (Kukar et al, 2008; Richter et al, 2010). However, this notion contradicts with the previous findings that several GSMs modulate the g-secretase-mediated cleavage of substrates other than APP (i.e., Notch); these GSMs affect the cleavage of APP mutated at the GXXXG motif too (Okochi et al, 2006; Page et al, 2010). Moreover, the activity of SPP, a protease homologous to g-secretase, also was affected by GSMs (Sato T et al, 2006). "
ABSTRACT: Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.The EMBO Journal 11/2011; 30(23):4815-24. DOI:10.1038/emboj.2011.372 · 10.75 Impact Factor
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- "The D6E10 antibody does not significantly cross-react with the m-apoE (Thal et al. 2007). For detection of Ab, an antibody raised against Ab 17–24 (4G8; Covance, Princeton, NJ, USA; 1/5,000) or a rabbit polyclonal antibody raised against Ab 1–42 [3,552, polyclonal rabbit, 1/1,000 (Page et al. 2010)] were used with formic acid pre-treatment. For staining astrocytes, an antibody raised against glial fibrillary acidic protein (GFAP; polyclonal rabbit; DAKO, Glostrup, Denmark; 1/1,000) was used. "
ABSTRACT: The deposition of amyloid-β protein (Aβ) in the brain is a hallmark of Alzheimer’s disease (AD). Apolipoprotein E (apoE) is involved in the clearance of Aβ from brain and the APOE ε4 allele is a major risk factor for sporadic AD. We have recently shown that apoE is drained into the perivascular space (PVS), where it co-localizes with Aβ. To further clarify the role of apoE in perivascular clearance of Aβ, we studied apoE-transgenic mice over-expressing human apoE4 either in astrocytes (GE4) or in neurons (TE4). These animals were crossbred with amyloid precursor protein (APP)-transgenic mice and with APP-presenilin-1 (APP-PS1) double transgenic mice. Using an antibody that specifically detects human apoE (h-apoE), we observed that astroglial expression of h-apoE in GE4 mice leads to its perivascular drainage, whereas neuronal expression in TE4 mice does not, indicating that neuron-derived apoE is usually not the subject of perivascular drainage. However, h-apoE was observed not only in the PVS of APP-GE4 and APP-PS1-GE4 mice, but also in that of APP-TE4 and APP-PS1-TE4 mice. In all these mouse lines, we found co-localization of neuron-derived h-apoE and Aβ in the PVS. Aβ and h-apoE were also found in the cytoplasm of perivascular astrocytes indicating that astrocytes take up the neuron-derived apoE bound to Aβ, presumably prior to its clearance into the PVS. The uptake of apoE–Aβ complexes into glial cells was further investigated in glioblastoma cells. It was mediated by α2macroglobulin receptor/low density lipoprotein receptor-related protein (LRP-1) and inhibited by adding receptor-associated protein (RAP). It results in endosomal Aβ accumulation within these cells. These results suggest that neuronal apoE–Aβ complexes, but not neuronal apoE alone, are substrates for LRP-1-mediated astroglial uptake, transcytosis, and subsequent perivascular drainage. Thus, the production of Aβ and its interaction with apoE lead to the pathological perivascular drainage of neuronal apoE and provide insight into the pathological interactions of Aβ with neuronal apoE metabolism. KeywordsAlzheimer’s disease–Astrocytes–Amyloid β-protein–Apolipoprotein E–Perivascular space–Neurons–Blood–brain barrierJournal of Neural Transmission 05/2011; 118(5):699-712. DOI:10.1007/s00702-010-0572-7 · 2.87 Impact Factor
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ABSTRACT: γ-Secretase generates the peptides of Alzheimer's disease, Aβ(40) and Aβ(42), by cleaving the amyloid precursor protein within its transmembrane domain. γ-Secretase also cleaves numerous other substrates, raising concerns about γ-secretase inhibitor off-target effects. Another important class of drugs, γ-secretase modulators, alter the cleavage site of γ-secretase on amyloid precursor protein, changing the Aβ(42)/Aβ(40) ratio, and are thus a promising therapeutic approach for Alzheimer's disease. However, the target for γ-secretase modulators is uncertain, with some data suggesting that they function on γ-secretase, whereas others support their binding to the amyloid precursor. In this paper we address this controversy by using a fluorescence resonance energy transfer-based assay to examine whether γ-secretase modulators alter Presenilin-1/γ-secretase conformation in intact cells in the absence of its natural substrates such as amyloid precursor protein and Notch. We report that the γ-secretase allosteric site is located within the γ-secretase complex, but substrate docking is needed for γ-secretase modulators to access this site.Nature Communications 11/2010; 1:130. DOI:10.1038/ncomms1129 · 10.74 Impact Factor