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FTY720/fingolimod, a sphingosine analogue, reduces amyloid-β production in neurons.

Takasugi N, Sasaki T, Ebinuma I, Osawa S, Isshiki H, Takeo K, Tomita T, Iwatsubo T - PLoS ONE (2013)

Bottom Line: Pharmacological analyses suggested that the mechanism of FTY720-mediated Aβ decrease in cells was independent of known downstream signaling pathways of S1PRs.Unexpectedly, 6-days treatment of APP transgenic mice with FTY720 resulted in a decrease in Aβ40, but an increase in Aβ42 levels in brains.These results suggest that S1PR modulators are novel type of regulators for Aβ metabolisms that are active in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Sphingosine-1-phosphate (S1P) is a pluripotent lipophilic mediator working as a ligand for G-protein coupled S1P receptors (S1PR), which is currently highlighted as a therapeutic target for autoimmune diseases including relapsing forms of multiple sclerosis. Sphingosine related compounds, FTY720 and KRP203 known as S1PR modulators, are phosphorylated by sphingosine kinase 2 (SphK2) to yield the active metabolites FTY720-P and KRP203-P, which work as functional antagonists for S1PRs. Here we report that FTY720 and KRP203 decreased production of Amyloid-β peptide (Aβ), a pathogenic proteins causative for Alzheimer disease (AD), in cultured neuronal cells. Pharmacological analyses suggested that the mechanism of FTY720-mediated Aβ decrease in cells was independent of known downstream signaling pathways of S1PRs. Unexpectedly, 6-days treatment of APP transgenic mice with FTY720 resulted in a decrease in Aβ40, but an increase in Aβ42 levels in brains. These results suggest that S1PR modulators are novel type of regulators for Aβ metabolisms that are active in vitro and in vivo.

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FTY720 decreased the γ-secretase-mediated cleavage of APP.SC100 were transiently transfected in N2a cells. After 24 hrs transfection, cells were treated with FTY720 or KRP203 for 24 hrs. (A) Levels of secreted human Aβ detected by human Aβ-specific ELISA (n = 4, mean ± SEM **P<0.01, ***P<0.001). (B) Immunoblotting analysis of secreted human Aβ separated by modified Tris/Tricin/8M Urea gel system. (C) Immunoblot analysis of APP CTFs including overexpressed SC100 and endogenous PS1 in FTY720-treated cell lysates. Quantification analysis of (C) for βCTF (D), αCTF (E) and AICD (F) (n = 4, mean ± SEM *P<0.05). (G) Immunoblot analysis of endogenous sAPPα and sAPPβ in the conditioned media of N2a cells.
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pone-0064050-g002: FTY720 decreased the γ-secretase-mediated cleavage of APP.SC100 were transiently transfected in N2a cells. After 24 hrs transfection, cells were treated with FTY720 or KRP203 for 24 hrs. (A) Levels of secreted human Aβ detected by human Aβ-specific ELISA (n = 4, mean ± SEM **P<0.01, ***P<0.001). (B) Immunoblotting analysis of secreted human Aβ separated by modified Tris/Tricin/8M Urea gel system. (C) Immunoblot analysis of APP CTFs including overexpressed SC100 and endogenous PS1 in FTY720-treated cell lysates. Quantification analysis of (C) for βCTF (D), αCTF (E) and AICD (F) (n = 4, mean ± SEM *P<0.05). (G) Immunoblot analysis of endogenous sAPPα and sAPPβ in the conditioned media of N2a cells.

Mentions: To further identify the molecular target of S1PR modulators, we analyzed their effect on N2a cells transiently expressing SC100, corresponding to βCTF of human APP that is a direct substrate of γ-secretase. SC100 is endoproteolyzed by γ-secretase at ε-site to release the intracellular domain (AICD), and resultant intramembrane stub is trimmed by carboxypeptidase-like activity of the γ-secretase at multiple γ-sites to generate Aβ. FTY720 and KRP203 decreased secretion of Aβ40 and Aβ42 from SC100 (Fig. 2A), suggesting that S1PR modulators affected the γ-secretase activity. Recently, small compounds that specifically lower Aβ42, and Aβ40 to a lesser extent, have been termed as γ-secretase modulators [6], [29]. However, FTY720 treatment decreased all Aβ species with different Aβ C-termini, including Aβ38, Aβ39, Aβ40 and Aβ42 (Fig. 2B). Concomitantly, a slight accumulation of αCTF (from endogenous mouse APP) and SC100 was observed (Fig. 2C–F), whereas the levels of sAPPα and sAPPβ products of α- or β-secretase-mediated APP cleavage of endogenous APP, respectively, were not altered by FTY720 treatment (Fig. 2G). Intriguingly, AICD production was not altered by FTY720 treatment (Fig. 2F) similarly to the processing of NΔE, a direct Notch substrate for γ-secretase (see Fig. 1D and E). Because both AICD and NICD are produced from ε-cleavage by γ-secretase, these data suggest that S1PR modulators specifically regulate the γ-cleavage irrespective of the substrate.


FTY720/fingolimod, a sphingosine analogue, reduces amyloid-β production in neurons.

Takasugi N, Sasaki T, Ebinuma I, Osawa S, Isshiki H, Takeo K, Tomita T, Iwatsubo T - PLoS ONE (2013)

FTY720 decreased the γ-secretase-mediated cleavage of APP.SC100 were transiently transfected in N2a cells. After 24 hrs transfection, cells were treated with FTY720 or KRP203 for 24 hrs. (A) Levels of secreted human Aβ detected by human Aβ-specific ELISA (n = 4, mean ± SEM **P<0.01, ***P<0.001). (B) Immunoblotting analysis of secreted human Aβ separated by modified Tris/Tricin/8M Urea gel system. (C) Immunoblot analysis of APP CTFs including overexpressed SC100 and endogenous PS1 in FTY720-treated cell lysates. Quantification analysis of (C) for βCTF (D), αCTF (E) and AICD (F) (n = 4, mean ± SEM *P<0.05). (G) Immunoblot analysis of endogenous sAPPα and sAPPβ in the conditioned media of N2a cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646787&req=5

pone-0064050-g002: FTY720 decreased the γ-secretase-mediated cleavage of APP.SC100 were transiently transfected in N2a cells. After 24 hrs transfection, cells were treated with FTY720 or KRP203 for 24 hrs. (A) Levels of secreted human Aβ detected by human Aβ-specific ELISA (n = 4, mean ± SEM **P<0.01, ***P<0.001). (B) Immunoblotting analysis of secreted human Aβ separated by modified Tris/Tricin/8M Urea gel system. (C) Immunoblot analysis of APP CTFs including overexpressed SC100 and endogenous PS1 in FTY720-treated cell lysates. Quantification analysis of (C) for βCTF (D), αCTF (E) and AICD (F) (n = 4, mean ± SEM *P<0.05). (G) Immunoblot analysis of endogenous sAPPα and sAPPβ in the conditioned media of N2a cells.
Mentions: To further identify the molecular target of S1PR modulators, we analyzed their effect on N2a cells transiently expressing SC100, corresponding to βCTF of human APP that is a direct substrate of γ-secretase. SC100 is endoproteolyzed by γ-secretase at ε-site to release the intracellular domain (AICD), and resultant intramembrane stub is trimmed by carboxypeptidase-like activity of the γ-secretase at multiple γ-sites to generate Aβ. FTY720 and KRP203 decreased secretion of Aβ40 and Aβ42 from SC100 (Fig. 2A), suggesting that S1PR modulators affected the γ-secretase activity. Recently, small compounds that specifically lower Aβ42, and Aβ40 to a lesser extent, have been termed as γ-secretase modulators [6], [29]. However, FTY720 treatment decreased all Aβ species with different Aβ C-termini, including Aβ38, Aβ39, Aβ40 and Aβ42 (Fig. 2B). Concomitantly, a slight accumulation of αCTF (from endogenous mouse APP) and SC100 was observed (Fig. 2C–F), whereas the levels of sAPPα and sAPPβ products of α- or β-secretase-mediated APP cleavage of endogenous APP, respectively, were not altered by FTY720 treatment (Fig. 2G). Intriguingly, AICD production was not altered by FTY720 treatment (Fig. 2F) similarly to the processing of NΔE, a direct Notch substrate for γ-secretase (see Fig. 1D and E). Because both AICD and NICD are produced from ε-cleavage by γ-secretase, these data suggest that S1PR modulators specifically regulate the γ-cleavage irrespective of the substrate.

Bottom Line: Pharmacological analyses suggested that the mechanism of FTY720-mediated Aβ decrease in cells was independent of known downstream signaling pathways of S1PRs.Unexpectedly, 6-days treatment of APP transgenic mice with FTY720 resulted in a decrease in Aβ40, but an increase in Aβ42 levels in brains.These results suggest that S1PR modulators are novel type of regulators for Aβ metabolisms that are active in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Sphingosine-1-phosphate (S1P) is a pluripotent lipophilic mediator working as a ligand for G-protein coupled S1P receptors (S1PR), which is currently highlighted as a therapeutic target for autoimmune diseases including relapsing forms of multiple sclerosis. Sphingosine related compounds, FTY720 and KRP203 known as S1PR modulators, are phosphorylated by sphingosine kinase 2 (SphK2) to yield the active metabolites FTY720-P and KRP203-P, which work as functional antagonists for S1PRs. Here we report that FTY720 and KRP203 decreased production of Amyloid-β peptide (Aβ), a pathogenic proteins causative for Alzheimer disease (AD), in cultured neuronal cells. Pharmacological analyses suggested that the mechanism of FTY720-mediated Aβ decrease in cells was independent of known downstream signaling pathways of S1PRs. Unexpectedly, 6-days treatment of APP transgenic mice with FTY720 resulted in a decrease in Aβ40, but an increase in Aβ42 levels in brains. These results suggest that S1PR modulators are novel type of regulators for Aβ metabolisms that are active in vitro and in vivo.

Show MeSH
Related in: MedlinePlus