Korean J Physiol Pharmacol.  2020 Jul;24(4):299-310. 10.4196/kjpp.2020.24.4.299.

Combination therapy with cilostazol, aripiprazole, and donepezil protects neuronal cells from β-amyloid neurotoxicity through synergistically enhanced SIRT1 expression

• Heo HJ
• Park SY
• Lee YS
• Shin HK
• Hong KW
• Kim CD

• KMID: 2503323
• DOI: http://doi.org/10.4196/kjpp.2020.24.4.299

Abstract

Alzheimer’s disease (AD) is a multi-faceted neurodegenerative disease. Thus, current therapeutic strategies require multitarget-drug combinations to treat or prevent the disease. At the present time, single drugs have proven to be inadequate in terms of addressing the multifactorial pathology of AD, and multitarget-directed drug design has not been successful. Based on these points of views, it is judged that combinatorial drug therapies that target several pathogenic factors may offer more attractive therapeutic options. Thus, we explored that the combination therapy with lower doses of cilostazol and aripiprazole with add-on donepezil (CAD) might have potential in the pathogenesis of AD. In the present study, we found the superior efficacies of donepezil add-on with combinatorial mixture of cilostazol plus aripiprazole in modulation of expression of AD-relevant genes: Aβ accumulation, GSK-3β, P300, acetylated tau, phosphorylated-tau levels, and activation of α-secretase/ADAM 10 through SIRT1 activation in the N2a Swe cells expressing human APP Swedish mutation (N2a Swe cells). We also assessed that CAD synergistically raised acetylcholine release and choline acetyltransferase (CHAT) expression that were declined by increased β-amyloid level in the activated N2a Swe cells. Consequently, CAD treatment synergistically increased neurite elongation and improved cell viability through activations of PI3K, BDNF, β-catenin and a7-nicotinic cholinergic receptors in neuronal cells in the presence of Aβ_1-42. This work endorses the possibility for efficient treatment of AD by supporting the synergistic therapeutic potential of donepezil add-on therapy in combination with lower doses of cilostazol and aripiprazole.

Keyword

Alzheimer’s disease; Aripiprazole; β-amyloid; Cilostazol; Donepezil

Figure

• 

  Fig. 1 Time-dependent increases in Aβ production and phosphorylation. (A) Time-dependent increases in Aβ production in mouse neuroblastoma neuro2A (N2a) Swe cells as determined by ELISA, represented as percentages of N2a cells cultured for 24 h as 100%. (B) Time-dependent increases in Aβ phosphorylated Tau (P-tau), acetylated tau (Ac-tau), P300, and GSK-3β P-Tyr-216 levels as determined by Western blot. Cells were cultured in 1% FBS for 3, 12, 24, or 48 h, respectively. (C) Quantitative results are expressed as the mean percentages ± SEMs with respect to N2a cells cultured for 24 h (control = 100%) from five experiments. *p < 0.05, **p < 0.01; ***p < 0.001 vs. N2a controls.

• 

  Fig. 2 Effects of donepezil (DNP), cilostazol + aripiprazole (CA), and CA + DNP (CAD). (A) Suppressive effects of 3 μM donepezil (DNP), 3 μM cilostazol + 3 μM aripiprazole (CA), and CA + 3 μM DNP (CAD) on Aβ production in neuro2A (N2a) Swe cells as determined by ELISA, are represented as percentages of N2a cells cultured for 24 h. (B) Representative Western blots of GSK-3β P-Tyr-216, phosphorylated tau (P-tau), P300, and acetylated tau (Ac-tau) expression levels in activated N2a Swe cells cultured for 24 h. Quantitative analysis of GSK-3β P-Tyr 216 (C), P-tau (D), P300 (E), and Ac-tau expression levels (F). Results are expressed as the means ± SEMs of percentages of vehicle values (100%) of activated N2a Swe cells from four experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. vehicle (veh); $p < 0.05, $$$p < 0.001 vs. DNP.

• 

  Fig. 3 Decreased ADAM10 expression in the activated neuro2A (N2a) Swe cells and its recovery. (A) Time-dependent decreases in ADAM10 protein levels when N2a Swe cells were cultured in 1% FBS-containing medium for 3, 12, 24, or 48 h. Means ± standard error of the mean (SEMs) are expressed as percentages of values of N2a cell cultured for 24 h (100%) from four experiments. **p < 0.01; ***p < 0.001 vs. values of N2a cells cultured for 24 h. (B) Synergistic upregulation of decreased ADAM10 expression in the N2a Swe cells (at 24 h after FBS depletion [control] = 100%) by 3 μM donepezil (DNP), 3 μM cilostazol + 3 μM aripiprazole (CA) and CA + DNP (CAD). Inhibition of CAD-stimulated ADAM10 expression by 10 μM TIMP (an ADAM10 inhibitor). Means ± SEMs are expressed as percentages of vehicle values (100%) of activated N2a Swe cells from four experiments. ##p < 0.01, ###p < 0.001 vs. vehicle (veh); $p < 0.05, $$$p < 0.001 vs. DNP; †††p < 0.001 vs. CAD alone.

• 

  Fig. 4 Significantly decreased α-secretase activity at 24 h after FBS depletion and concentration-dependent recovery by cilostazol. (A), 3 μM cilostazol (C3) + 1, 3, 10 μM donepezil (D1, D3, D10) (B), 3 μM cilostazol (C3) + 1, 3, 10 μM aripiprazole (A1, A3, A10) (C). (D) Synergistic upregulation of α-secretase activity in the neuro2A (N2a) Swe cells by 3 μM donepezil (DNP) alone, 3 μM cilostazol + 3 μM aripiprazole (CA), CAD, and blocking of CAD-stimulated α-secretase activity by 10 μM Rp-cAMPS, 20 μM sirtinol and 10 μM TIMP, respectively. Means ± SEMs are expressed as percentages of control values (100%) of N2a cells (with 10% FBS) from 4–8 experiments. **p < 0.01, ***p < 0.001 vs. control; #p < 0.01, ###p < 0.001 vs. vehicle (veh); $$p < 0.01, $$$p < 0.001 vs. DNP; †††p < 0.001 vs. CAD alone. ϕϕϕp < 0.001 vs. CA.

• 

  Fig. 5 Drug effects on SIRT1 expression and deacetylase activity in the activated neuro2A (N2a) Swe cells. (A) Upregulation of SIRT1 expression in the N2a Swe cells by 3 μM donepezil (DNP), 3 μM cilostazol + 3 μM aripiprazole (CA) and CA + 3 μM DNP (CAD) and blocking of CAD-stimulated SIRT1 expression by 20 μM Sirtinol. Means ± standard error of the mean (SEMs) are expressed as percentages of vehicle (veh = 100%) of activated N2a Swe cells from 5 experiments. (B) Significantly decreased SIRT1 deacetylase activity at 24 h after 1% FBS medium and synergistic upregulation over the control level by drugs and blocking of CAD-stimulated SIRT1 expression by 20 μM sirtinol. (C) Synergistic upregulation of nuclear P-CREB Ser133 levels by DNP, CA, CAD, and blocking of CAD-stimulated intranuclear P-CREB Ser133 levels by 30 μM of LY294002. Means ± SEMs are expressed as percentages of control (100%) of N2a Swe cells (10% FBS) from 4 experiments. *p < 0.05 vs. control; ##p < 0.01, ###p < 0.001 vs. vehicle (veh); $p < 0.05, $$p < 0.01, $$$p < 0.001 vs. DNP; Ψp < 0.05 vs. CA; †p < 0.05, ††p < 0.01, †††p < 0.001 vs. CAD alone.

• 

  Fig. 6 Drug effects on acetylcholine (ACh) release and expression of choline acetyltransferase (CHAT) in the activated neuro2A (N2a) Swe cells. (A) Significant upregulation of decreased ACh release into the culture medium containing 1% FBS with N2a Swe cells (24 h) by 3 μM donepezil (DNP), 3 μM cilostazol + 3 μM aripiprazole (CA) and CA + 3 μM DNP (CAD). (B) Significant upregulation of CHAT expression by CAD, which was blocked by 10 nM MLA (methyllycaconitine). Means ± SEMs are expressed as percentages of control (ACh; 0.15 ± 0.03 pg/ml = 100%) of N2a Swe cells (10% FBS) from 5 experiments. *p < 0.05, ***p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. vehicle; $$p < 0.01 vs. DNP; Ψp < 0.05 vs. CA; †††p < 0.001 vs. CAD alone.

• 

  Fig. 7 Representative microscopic features (A) and their quantitative analyses (B). The neurite elongation of the HT22 cells was markedly decreased by 3 μM Aβ1-42, which was recovered over the control level by treatment with CA and synergistically increased by CAD, but not by 3 μm donepezil (DNP) alone. Blocking of CAD-stimulated neurite lengths by LY294002 (30 μM), K252A (100 nM), imatinib (10 μM) and methyllycaconitine (MLA) (10 nM), respectively. Results are expressed as means ± SEMs of neurite length (μm) in HT22 cells from 7–14 experiments. ***p < 0.001 vs. control (con); ###p < 0.001 vs. vehicle (veh); $$$p < 0.001 vs. DNP; ΨΨΨp < 0.001 vs. CA; †††p < 0.001 vs. CAD alone.

• 

  Fig. 8 Effects of drugs on the cell viability of neuro2A (N2a) cells. Cells were pretreated with CAD (3 μM donepezil + 3 μM cilostazol + 3 μM aripiprazole) for 2 h before addition of Aβ1-42 (20 μM) and then cultured forward for 24 h in the absence and presence of 30 μM of LY294002, 100 nM of K252A, 10 μM of imatinib and 10 nM of methyllycaconitine (MLA), respectively. Results are expressed as means ± SEMs of cell viability from 5 experiments. ***p < 0.001 vs. control; #p < 0.001 vs. vehicle; ††p < 0.01, †††p < 0.001 vs. CAD alone.

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