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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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SphK2 activity is required for FTY720 mediated decrease of Aβ secretion.(A) N2a cells were transfected with siRNA against murine SphK2. After 48 hrs transfection of siRNA, levels of SphK2 was detected by immunoblotting (upper panel) and quantified (lower graph n = 3, mean ± SEM). (B) After 48 hrs transfection of siRNA, cells were treated with FTY720 for 24 hrs. Levels of secreted Aβ were quantified by ELISA (n = 3, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or siRNA against SphK2 (indicated by line)). One-way ANOVA with Tukey's post hoc test for individual treatment differences was used for statistical analysis. (C, D) N2a cells were transiently transfected with LacZ, wild-type (WT) or dominant negative mutant (G243D) SphK2. After 24 hrs transfection, cells were treated with FTY720 for 24 hrs. (C) Levels of secreted Aβ (n = 4, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or SphK2 (indicated by line)). (D) The inhibitory efficiency of FTY720 on Aβ secretion compared with DMSO treatment in each transfection of (C). Secreted Aβ levels of FTY720 were standardized by vehicle control in each group (mean ± SEM; **P<0.01).
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pone-0064050-g003: SphK2 activity is required for FTY720 mediated decrease of Aβ secretion.(A) N2a cells were transfected with siRNA against murine SphK2. After 48 hrs transfection of siRNA, levels of SphK2 was detected by immunoblotting (upper panel) and quantified (lower graph n = 3, mean ± SEM). (B) After 48 hrs transfection of siRNA, cells were treated with FTY720 for 24 hrs. Levels of secreted Aβ were quantified by ELISA (n = 3, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or siRNA against SphK2 (indicated by line)). One-way ANOVA with Tukey's post hoc test for individual treatment differences was used for statistical analysis. (C, D) N2a cells were transiently transfected with LacZ, wild-type (WT) or dominant negative mutant (G243D) SphK2. After 24 hrs transfection, cells were treated with FTY720 for 24 hrs. (C) Levels of secreted Aβ (n = 4, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or SphK2 (indicated by line)). (D) The inhibitory efficiency of FTY720 on Aβ secretion compared with DMSO treatment in each transfection of (C). Secreted Aβ levels of FTY720 were standardized by vehicle control in each group (mean ± SEM; **P<0.01).

Mentions: Phosphorylation of FTY720 by SphK2 yields the active metabolite, FTY720-phosphate (FTY720-P), which is known as a potent agonist of the S1P receptors (Fig. 1A). To determine whether FTY720-P is involved in the regulation of Aβ production, we treated N2a cells with FTY720 after RNAi knock-down of the endogenous expression of SphK2. We observed a ∼60% of decrease in SphK2 expression after siRNA treatment (Fig 3A). As reported previously, knockdown of SphK2 decreased Aβ secretion [22]. However, additional decrease was not observed in FTY720-treated SphK2 knockdown cells, suggesting that SphK2 is required for lowering Aβ secretion by FTY720 (Fig. 3B). Next, we examined the effects of overexpression of SphK2 or its dominant negative mutant (G243D) in N2a cells. As reported previously, overexpression of SphK2 increased Aβ production [22] (Fig. 3C). Intriguingly, FTY720 treatment significantly decreased Aβ secretion from N2a cells that overexpress wild-type (WT) SphK2, but not dominant negative mutant, to levels lower than those of untransfected cells treated by FTY720. Quantitative comparison of the inhibitory effects of FTY720 revealed that an increase in SphK2 activity significantly sensitized N2a cells to the inhibitory effect of FTY720 on Aβ production (Fig. 3D). These data strongly suggest SphK2 activity is involved in the mechanism of action of FTY720 to lower Aβ production, raising the possibility that FTY720-P is the bona fide regulator of the γ-secretase activities.


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)

SphK2 activity is required for FTY720 mediated decrease of Aβ secretion.(A) N2a cells were transfected with siRNA against murine SphK2. After 48 hrs transfection of siRNA, levels of SphK2 was detected by immunoblotting (upper panel) and quantified (lower graph n = 3, mean ± SEM). (B) After 48 hrs transfection of siRNA, cells were treated with FTY720 for 24 hrs. Levels of secreted Aβ were quantified by ELISA (n = 3, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or siRNA against SphK2 (indicated by line)). One-way ANOVA with Tukey's post hoc test for individual treatment differences was used for statistical analysis. (C, D) N2a cells were transiently transfected with LacZ, wild-type (WT) or dominant negative mutant (G243D) SphK2. After 24 hrs transfection, cells were treated with FTY720 for 24 hrs. (C) Levels of secreted Aβ (n = 4, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or SphK2 (indicated by line)). (D) The inhibitory efficiency of FTY720 on Aβ secretion compared with DMSO treatment in each transfection of (C). Secreted Aβ levels of FTY720 were standardized by vehicle control in each group (mean ± SEM; **P<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646787&req=5

pone-0064050-g003: SphK2 activity is required for FTY720 mediated decrease of Aβ secretion.(A) N2a cells were transfected with siRNA against murine SphK2. After 48 hrs transfection of siRNA, levels of SphK2 was detected by immunoblotting (upper panel) and quantified (lower graph n = 3, mean ± SEM). (B) After 48 hrs transfection of siRNA, cells were treated with FTY720 for 24 hrs. Levels of secreted Aβ were quantified by ELISA (n = 3, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or siRNA against SphK2 (indicated by line)). One-way ANOVA with Tukey's post hoc test for individual treatment differences was used for statistical analysis. (C, D) N2a cells were transiently transfected with LacZ, wild-type (WT) or dominant negative mutant (G243D) SphK2. After 24 hrs transfection, cells were treated with FTY720 for 24 hrs. (C) Levels of secreted Aβ (n = 4, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or SphK2 (indicated by line)). (D) The inhibitory efficiency of FTY720 on Aβ secretion compared with DMSO treatment in each transfection of (C). Secreted Aβ levels of FTY720 were standardized by vehicle control in each group (mean ± SEM; **P<0.01).
Mentions: Phosphorylation of FTY720 by SphK2 yields the active metabolite, FTY720-phosphate (FTY720-P), which is known as a potent agonist of the S1P receptors (Fig. 1A). To determine whether FTY720-P is involved in the regulation of Aβ production, we treated N2a cells with FTY720 after RNAi knock-down of the endogenous expression of SphK2. We observed a ∼60% of decrease in SphK2 expression after siRNA treatment (Fig 3A). As reported previously, knockdown of SphK2 decreased Aβ secretion [22]. However, additional decrease was not observed in FTY720-treated SphK2 knockdown cells, suggesting that SphK2 is required for lowering Aβ secretion by FTY720 (Fig. 3B). Next, we examined the effects of overexpression of SphK2 or its dominant negative mutant (G243D) in N2a cells. As reported previously, overexpression of SphK2 increased Aβ production [22] (Fig. 3C). Intriguingly, FTY720 treatment significantly decreased Aβ secretion from N2a cells that overexpress wild-type (WT) SphK2, but not dominant negative mutant, to levels lower than those of untransfected cells treated by FTY720. Quantitative comparison of the inhibitory effects of FTY720 revealed that an increase in SphK2 activity significantly sensitized N2a cells to the inhibitory effect of FTY720 on Aβ production (Fig. 3D). These data strongly suggest SphK2 activity is involved in the mechanism of action of FTY720 to lower Aβ production, raising the possibility that FTY720-P is the bona fide regulator of the γ-secretase activities.

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