Specific Triazine Herbicides Induce Amyloid-β42 Production
Erik PorteliusÉmilie DurieuMarion BodinMorgane CamJosef PanneeCharlotte LeuxeAloı̈se MabondzoNassima OumataHervé GalonsJung Yeol LeeYoung‐Tae ChangKathrin StüberPhilipp KochGaëlle FontaineMarie‐Claude PotierAntigoni ManousopoulouSpiros D. GarbisAdrian CovaciDebby Van DamPeter Paul De DeynFrank KargMarc FlajoletChiori OmoriSaori HataToshiharu SuzukiKaj BlennowHenrik ZetterbergLaurent Meijer
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Abstract:
Proteolytic cleavage of the amyloid-β protein precursor (AβPP) by secretases leads to extracellular release of amyloid-β (Aβ) peptides. Increased production of Aβ42 over Aβ40 and aggregation into oligomers and plaques constitute an Alzheimer's disease (AD) hallmark. Identifying products of the 'hum an chemical exposome' (HCE) able to induce Aβ42 production may be a key to understanding some of the initiating causes of AD and to generate non-genetic, chemically-induced AD animal models. A cell model was used to screen HCE libraries for Aβ42 inducers. Out of 3500+ compounds, six triazine herbicides were found that induced a β- and γ-secretases-dependent, 2–10 fold increase in the production of extracellular Aβ42 in various cell lines, primary neuronal cells, and neurons differentiated from human-induced pluripotent stem cells (iPSCs). Immunoprecipitation/mass spectrometry analyses show enhanced production of Aβ peptides cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and lower, a characteristic of AD. Neurons derived from iPSCs obtained from a familial AD (FAD) patient (AβPP K724N) produced more Aβ42 versus Aβ40 than neurons derived from healthy controls iPSCs (AβPP WT). Triazines enhanced Aβ42 production in both control and AD iPSCs-derived neurons. Triazines also shifted the cleavage pattern of alcadeinα, another γ-secretase substrate, suggesting a direct effect of triazines on γ-secretase activity. In conclusion, several widely used triazines enhance the production of toxic, aggregation prone Aβ42/Aβ43 amyloids, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in late-onset AD.Keywords:
Cleavage (geology)
Amyloid (mycology)
Alpha secretase
Alpha secretase
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Cleavage (geology)
Ectodomain
Immunoprecipitation
Amyloid (mycology)
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Amyloid-β peptide (AβP) that accumulates in the Alzheimer's diseased brain is derived from proteolytic processing of the amyloid precursor protein (APP) by means of β- and γ-secretases. The β-secretase APP cleaving enzyme (BACE), which generates the N terminus of AβP, has become a target of intense research aimed at blocking the enzyme activity, thus reducing AβP and, subsequently, plaque formation. The search for specific inhibitors of β-secretase activity as a possible treatment for Alzheimer's disease intensified with the discovery that BACE may be involved in processing other non-APP substrates. The presence of the APP–BACE complex in early endosomes highlights the cell surface as a potential therapeutic target, suggesting that interference in APP–BACE interaction at the cell surface may affect amyloid-β production. We present here a unique approach to inhibit AβP production by means of antibodies against the β-secretase cleavage site of APP. These antibodies were found to bind human APP overexpressed by CHO cells, and the formed immunocomplex was visualized in the early endosomes. Indeed, blocking of the β-secretase site by these antibodies interfered with BACE activity and inhibited both intracellular and extracellular AβP formation in these cells.
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The amyloid precursor family of proteins are of considerable interest, both because of their role in Alzheimer’s disease pathogenesis and because of their normal physiological functions. In mammals, the amyloid precursor protein (APP) has two homologs, amyloid precursor‐like protein (APLP) 1 and APLP2. All three proteins undergo ectodomain shedding and regulated intramembrane proteolysis, and important functions have been attributed to the full‐length proteins, shed ectodomains, C‐terminal fragments and intracellular domains (ICDs). One of the proteases that is known to cleave APP and that is essential for generation of the amyloid β‐protein is the β‐site APP‐cleaving enzyme 1 (BACE1). Here, we investigated the effects of genetic manipulation of BACE1 on the processing of the APP family of proteins. BACE1 expression regulated the levels and species of full‐length APLP1, APP and APLP2, of their shed ectodomains, and of their membrane‐bound C‐terminal fragments. In particular, APP processing appears to be tightly regulated, with changes in β‐cleaved APPs (APPsβ) being compensated for by changes in α‐cleaved APPs (APPsα). In contrast, the total levels of soluble cleaved APLP1 and APLP2 species were less tightly regulated, and fluctuated with BACE1 expression. Importantly, the production of ICDs for all three proteins was not decreased by loss of BACE1 activity. These results indicate that BACE1 is involved in regulating ectodomain shedding, maturation and trafficking of the APP family of proteins. Consequently, whereas inhibition of BACE1 is unlikely to adversely affect potential ICD‐mediated signaling, it may alter other important facets of amyloid precursor‐like protein/APP biology.
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Neurotoxic amyloid β-peptides are thought to be a causative agent of Alzheimer's disease in humans. The production of amyloid β-peptides from amyloid precursor protein (APP) could be diminished by enhancing α-processing; however, the physical interactions between APP and α-secretases are not well understood. In this study, we employed super-resolution light microscopy to examine in cell-free plasma membranes the abundance and association of APP and α-secretases ADAM10 (a disintegrin and metalloproteinase) and ADAM17. We found that both secretase molecules localize similarly closely to APP (within ≤50 nm). However, when cross-linking APP with antibodies directed against the GFP tag of APP, in confocal microscopy, we observed that only ADAM10 coaggregated with APP. Furthermore, we mapped the involved protein domain by using APP variants with an exchanged transmembrane segment or lacking cytoplasmic/extracellular domains. We identified that the transmembrane domain of APP is required for association with α-secretases and, as analyzed by Western blot, for α-processing. We propose that the transmembrane domain of APP interacts either directly or indirectly with ADAM10, but not with ADAM17, explaining the dominant role of ADAM10 in α-processing of APP. Further understanding of this interaction may facilitate the development of a therapeutic strategy based on promoting APP cleavage by α-secretases.
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Amyloid-β precursor protein (APP), a type I membrane protein, is physiologically processed by α- or β-secretases that cleave APP N-terminal to the transmembrane region. Extracellular α-/β-cleavage of APP generates a large secreted N-terminal fragment, and a smaller cellular C-terminal fragment. Subsequent γ-secretase cleavage in the transmembrane region of the C-terminal fragment induces secretion of small extracellular peptides, including Aβ 40 and Aβ 42 , which are instrumental in the pathogenesis of Alzheimer's disease, and intracellular release of a cytoplasmic tail fragment. Although APP resembles a cell-surface receptor, no functionally active extracellular ligand for APP that might regulate its proteolytic processing has been described. We now show that F-spondin, a secreted signaling molecule implicated in neuronal development and repair, binds to the conserved central extracellular domain of APP and inhibits β-secretase cleavage of APP. Our data indicate that F-spondin may be an endogenous regulator of APP cleavage, and suggest that the extracellular domains of APP are potential drug targets for interfering with β-secretase cleavage.
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P3 peptide
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Alzheimer-disease-associated β-amyloid (Aβ) is produced by sequential endoproteolysis of β-amyloid protein precursor (βAPP): the extracellular portion is shed by cleavage in the juxtamembrane region by β-amyloid-cleaving enzyme (BACE)/β-secretase, after which it is cleaved by presenilin (PS)/γ-secretase near the middle of the transmembrane domain. Thus, inhibition of either of the secretases reduces Aβ generation and is a fundamental strategy for the development of drugs to prevent Alzheimer disease. However, it is not clear how small compounds reduce Aβ production without inhibition of the secretases. Such compounds are expected to avoid some of the side effects of secretase inhibitors. Here, we report that destruxin E (Dx-E), a natural cyclic hexadepsipeptide, reduces Aβ generation without affecting BACE or PS/γ-secretase activity. In agreement with this, Dx-E did not inhibit Notch signaling. We found that Dx-E decreases colocalization of BACE1 and βAPP, which reduces β-cleavage of βAPP. Therefore, the data demonstrate that Dx-E represents a novel Aβ-reducing process which could have fewer side effects than secretase inhibitors.
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Alpha secretase
Gamma secretase
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P3 peptide
Cleavage (geology)
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Amyloid beta-peptide (Abeta) is implicated as the major causative agent in Alzheimer's disease (AD). Abeta is produced by the processing of the amyloid precursor protein (APP) by BACE1 (beta-secretase) and gamma-secretase. Many inhibitors have been developed for the secretases. However, the inhibitors will interfere with the processing of not only APP but also of other secretase substrates. In this study, we describe the development of inhibitors that prevent production of Abeta by specific binding to the beta-cleavage site of APP. We used the hydropathic complementarity (HC) approach for the design of short peptide inhibitors. Some of the HC peptides were bound to the substrate peptide (Sub W) corresponding to the beta-cleavage site of APP and blocked its cleavage by recombinant human BACE1 (rhBACE1) in vitro. In addition, HC peptides specifically inhibited the cleavage of Sub W, and not affecting other BACE1 substrates. Chemical modification allowed an HC peptide (CIQIHF) to inhibit the processing of APP as well as the production of Abeta in the treated cells. Such novel APP-specific inhibitors will provide opportunity for the development of drugs that can be used for the prevention and treatment of AD with minimal side effects.
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P3 peptide
Cleavage (geology)
Amyloid beta
Protein precursor
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The Amyloid Precursor Protein (APP) undergoes sequential proteolytic cleavages through the action of beta- and gamma-secretase, which result in the generation of toxic beta-amyloid (Abeta) peptides and a C-terminal fragment consisting of the intracellular domain of APP (AICD). Mutations leading to increased APP levels or alterations in APP cleavage cause familial Alzheimer's disease (AD). Thus, identification of factors that regulate APP steady state levels and/or APP cleavage by gamma-secretase is likely to provide insight into AD pathogenesis. Here, using transgenic flies that act as reporters for endogenous gamma-secretase activity and/or APP levels (GAMAREP), and for the APP intracellular domain (AICDREP), we identified mutations in X11L and ubiquilin (ubqn) as genetic modifiers of APP. Human homologs of both X11L (X11/Mint) and Ubqn (UBQLN1) have been implicated in AD pathogenesis. In contrast to previous reports, we show that overexpression of X11L or human X11 does not alter gamma-secretase cleavage of APP or Notch, another gamma-secretase substrate. Instead, expression of either X11L or human X11 regulates APP at the level of the AICD, and this activity requires the phosphotyrosine binding (PTB) domain of X11. In contrast, Ubqn regulates the levels of APP: loss of ubqn function leads to a decrease in the steady state levels of APP, while increased ubqn expression results in an increase in APP levels. Ubqn physically binds to APP, an interaction that depends on its ubiquitin-associated (UBA) domain, suggesting that direct physical interactions may underlie Ubqn-dependent regulation of APP. Together, our studies identify X11L and Ubqn as in vivo regulators of APP. Since increased expression of X11 attenuates Abeta production and/or secretion in APP transgenic mice, but does not act on gamma-secretase directly, X11 may represent an attractive therapeutic target for AD.
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Abeta is the major component of amyloid in the brain in Alzheimer's disease and is derived from an amyloid precursor protein (APP) by the sequential proteolytic processing of two putative proteases, called beta- and gamma-secretase. To clarify the mechanism of gamma-secretase processing, we created constructs contained the C-terminal domain of APP and analyzed the processing in COS-1 cells. We found that C-terminal fragments (CTFs) containing a short extra N-terminal region before the beta-secretase cleavage site were directly processed at gamma-secretase cleavage site. This suggests that gamma-secretase cleavage occurs independently and is not dependent on alpha- and beta-secretase cleavage.
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Alpha secretase
Amyloid (mycology)
Gamma secretase
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