Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

Hye Rin Lee,Ki Whan Hong,Hye Young Kim,Hwa Kyoung Shin,Sun Sik Bae,Won Suk Lee,Chi Dae Kim,So Youn Park,Byung Yong Rhim,Stephen D Ginsberg

doi:10.1371/journal.pone.0134486

Hye Rin Lee, Ki Whan Hong + Show 8 more

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https://doi.org/10.1371/journal.pone.0134486

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Journal: PloS one	Publication Date: Aug 5, 2015
Citations: 34	License type: CC BY 4.0

Affiliation: Pusan National University

Abstract

Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer’s disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1–42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10–30 μM) or resveratrol (20 μM) prevented these Aβ1–42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

Highlights

Alzheimer’s disease (AD) is characterized by extracellular amyloid β (Aβ)-containing plaques and intracellular neurofibrillary tangles (NFTs) consisting of aggregated phosphorylated-tau, and is accompanied by synaptic and neuronal failure and cognitive deficits [1]
Time-dependent decreases in beclin-1, autophagy-related protein5 (Atg5), and SIRT1 expressions in N2a cells in response to exogenous Aβ 1–42 and cilostazol effects
To determine whether Aβ1–42 affects beclin-1, Atg5, and SIRT1 levels, we examined their protein expressions in N2a cells

Summary

Introduction

Alzheimer’s disease (AD) is characterized by extracellular amyloid β (Aβ)-containing plaques and intracellular neurofibrillary tangles (NFTs) consisting of aggregated phosphorylated-tau, and is accompanied by synaptic and neuronal failure and cognitive deficits [1]. Aβ and amyloid precursor protein (APP) C-terminal fragments (CTFβ) contribute to the pathology of AD and exhibit neurotoxic properties through multiple pathways [2]. Failure to regulate the production and clearance of Aβ increases Aβ levels, which leads to neurotoxicity and contributes to the pathogenesis of AD [3]. An intracellular bulk degradation process of cellular constituents, has been reported to be highly efficient in healthy neurons and to protect them from Aβ-induced cytotoxicity [4, 5, 6], which is indicative of the neuroprotective role of autophagy against cytotoxic proteins in AD.

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