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.
"The brains were frozen and 25 μm thick coronal frozen sections were cut with a sliding microtome (SM200R, Leica Biosystems, Wetzlar, Germany). Immunofluorescent staining was performed on every tenth section with the anti-Aβ antibody 3552 (; 1:3,000, overnight at 4 °C; anti-rabbit secondary antibodies (Invitrogen, Life Technologies, Paisley, UK): Alexa555, 1:1,000 1 h at room temperature or Alexa488, 1:250, 1 h at room temperature) or anti-Aβ antibody 4G8 (Covance, 1:150, overnight at 4 °C; anti-mouse secondary antibody Alexa488 (Invitrogen), 1:250, 1 h at room temperature). Omission of primary antibodies showed no detectable staining. "
[Show abstract][Hide abstract] ABSTRACT: Amyloid-β (Aβ) plaque deposition plays a central role in the pathogenesis of Alzheimer’s disease (AD). Post-mortem analysis of plaque development in mouse models of AD revealed that plaques are initially small, but then increase in size and become more numerous with age. There is evidence that plaques can grow uniformly over time; however, a complementary hypothesis of plaque development is that small plaques cluster and grow together thereby forming larger plaques. To investigate the latter hypothesis, we studied plaque formation in APPPS1 mice using in vivo two-photon microscopy and immunohistochemical analysis. We used sequential pre- and post-mortem staining techniques to label plaques at different stages of development and to detect newly emerged plaques. Post-mortem analysis revealed that a subset (22 %) of newly formed plaques appeared very close (<40 μm) to pre-existing plaques and that many close plaques (25 %) that were initially separate merged over time to form one single large plaque. Our results suggest that small plaques can cluster together, thus forming larger plaques as a complementary mechanism to simple uniform plaque growth from a single initial plaque. This study deepens our understanding of Aβ deposition and demonstrates that there are multiple mechanisms at play in plaque development.
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The online version of this article (doi:10.1007/s00401-013-1137-2) contains supplementary material, which is available to authorized users.
"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). "
[Show abstract][Hide abstract] 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 · 10.43 Impact Factor
"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. "
[Show abstract][Hide abstract] 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 barrier
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