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. 2019 May:125:123-134.
doi: 10.1016/j.nbd.2019.01.020. Epub 2019 Jan 30.

Multi-faceted therapeutic strategy for treatment of Alzheimer's disease by concurrent administration of etodolac and α-tocopherol

Khaled H Elfakhri  1 , Ihab M Abdallah  2 , Andrew D Brannen  2 , Amal Kaddoumi  3
Affiliations

Multi-faceted therapeutic strategy for treatment of Alzheimer's disease by concurrent administration of etodolac and α-tocopherol

Khaled H Elfakhri et al. Neurobiol Dis. 2019 May.

Abstract

Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple dysfunctional pathways. Therefore, a sophisticated treatment strategy that simultaneously targets multiple brain cell types and disease pathways could be advantageous for effective intervention. To elucidate an effective treatment, we developed an in vitro high-throughput screening (HTS) assay to evaluate candidate drugs for their ability to enhance the integrity of the blood-brain barrier (BBB) and improve clearance of amyloid-β (Aβ) using a cell-based BBB model. Results from HTS identified etodolac and α-tocopherol as promising drugs for further investigation. Both drugs were tested separately and in combination for the purpose of targeting multiple pathways including neuroinflammation and oxidative stress. In vitro studies assessed the effects of etodolac and α-tocopherol individually and collectively for BBB integrity and Aβ transport, synaptic markers and Aβ production in APP-transfected neuronal cells, as well as effects on inflammation and oxidative stress in astrocytes. Transgenic 5XFAD mice were used to translate in vitro results of etodolac and α-tocopherol independently and with concurrent administration. Compared to either drug alone, the combination significantly enhanced the BBB function, decreased total Aβ load correlated with increased expression of major transport proteins, promoted APP processing towards the neuroprotective and non-amyloidogenic pathway, induced synaptic markers expression, and significantly reduced neuroinflammation and oxidative stress both in vitro and in vivo. Collective findings demonstrated the combination produced mixed interaction showing additive, less than additive or synergistic effects on the evaluated markers. In conclusion, this study highlights the significance of combination therapy to simultaneously target multiple disease pathways, and suggest the repurposing and combination of etodolac and α-tocopherol as a novel therapeutic strategy against AD.

Keywords: Alzheimer's disease; Amyloid-β; Blood-brain barrier; Combination therapy; Neuroinflammation; Oxidative stress.

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Conflict of interest statement

Conflicts of interest

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Combination (COMB) treatment significantly enhanced BBB intactness compared to vehicle (control = CTRL), and etodolac (ETO) and/or α-tocopherol (Tph) treated groups. (a) In vitro concentration-dependent effect of ETO, Tph, and COMB on permeation of the paracellular permeability marker 14C-inulin across a bEnd3 cell-based BBB model. Data represented as mean ± SD of 3–4 independent experiments with n=4 wells/treatment/experiment. (b) Representative brain sections of 5XFAD mice treated with ETO, Tph, and COMB stained with anti-IgG to quantify endogenous IgG extravasation (green) and anti-collagen antibody to resolve microvessels (red) in cortex and hippocampus regions with quantitative analysis of IgG optical density. Scale bar, 50 μm. Data represented as mean ± SEM of n=5 mice per treatment and n=9 mice for control group. ns = not significant; ** p < 0.01, *** p < 0.001 compared to CTRL; # p < 0.05, ## p < 0.01, ### p < 0.001represent COMB compared to ETO alone; @@ p < 0.01, @@@ p < 0.001represent COMB compared to Tph alone.
Figure 2.
Figure 2.
Combination (COMB) treatment significantly increased tight junction proteins expression in vitro in bEnd3 cells and in vivo in isolated mice brain microvessels compared to vehicle treated groups (control = CTRL), and etodolac (ETO) and/or α-tocopherol (Tph). (a) Representative Western blot and densitometry analysis of ZO-1 and claudin-5 expression in bEnd3 cells. Cells were treated with 10μM of each compound for 24 h. Data represented as mean ± SD of 3 independent experiments with n=3 dishes/treatment/experiment. (b) Representative Western blot and densitometry analysis of ZO-1 and claudin-5 in vivo from microvessels isolated from 5XFAD mice brains. Microvessels were isolated from the homogenate of the right hemisphere of mice brains (n=5–6 mice). Mice were treated with ETO, Tph or COMB at 10 mg/kg/day each for 30 days. Data represented as mean ± SEM of n=5–6 mice per group. ns = not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CTRL; # p < 0.05, ## p < 0.01 represent COMB compared to ETO alone; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001represent COMB compared to Tph alone.
Figure 3.
Figure 3.
Combination (COMB) treatment differentially and significantly altered Aβ transport across the cell-based BBB model associated with increased expression of Aβ major transport proteins in vitro in bEnd3 cells, and in vivo in isolated mice brain microvessels compared to control (CTRL), and etodolac (ETO) and/or α-tocopherol (Tph). (a) Concentration-dependent effects of ETO, Tph and COMB on the transport of 125I-Aβ40 across bEnd3 cells-based BBB model. Data represented as mean ± SD of 3 independent experiments with n=4 wells/treatment/experiment. (b) Representative Western blot and densitometry analysis of P-gp and LRP1 expressions in bEnd3 cells. Cells were treated with 10μM of each compound for 24 h. Data represented as mean ± SD of 3 independent experiments with n=3 dishes/treatment group/experiment. (c) Representative Western blot and densitometry analysis of P-gp and LRP1 expressions in vivo from microvessels isolated from 5XFAD mice brains. Microvessels were isolated from the homogenate of the right hemisphere of mice brains (n=5–6 mice). Mice were treated with ETO, Tph or COMB at 10 mg/kg/day each for 30 days. Data represented as mean ± SEM of n=5–6 mice per group. ns = not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CTRL; # p < 0.05, ### p < 0.001represent COMB compared to ETO alone; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001represent COMB compared to Tph alone.
Figure 4.
Figure 4.
Combination (COMB) treatment significantly reduced brain Aβ levels. (a) Quantitative analysis of Aβ40 and Aβ42 levels in mice brains homogenate as measured by ELISA (n=5–6 mice per group). (b) Representative brain sections of 5XFAD mice treated with etodolac (ETO), α-tocopherol (Tph), and COMB stained with 6E10 antibody to quantify total Aβ load (green) and anti-collagen antibody to detect microvessels (red), and their optical density quantitation in both brain regions, cortex and hippocampus (n=9 mice for control group and n=5 mice for treatment groups). Scale bar, 50 μm. Mice were treated with ETO, Tph or COMB at 10 mg/kg/day each for 30 days. Data are presented as mean ± SEM. ns = not significant; ** p < 0.01, *** p < 0.001 compared to CTRL; # p < 0.05, ## p < 0.01, ### p < 0.001represent COMB compared to ETO alone; @@@ p < 0.001represent COMB compared to Tph alone.
Figure 5.
Figure 5.
Combination (COMB) treatment significantly reduced neuroinflammation and oxidative stress biomarkers. (a) Effect of etodolac (ETO, 10 μM), α-tocopherol (Tph, 10 μM), and COMB treatments on COX2, PGE2, and NO levels in CCF-STTG1 astrocyte cells treated or not with IL-1β (10 ng/ml) for 24 h. Baseline represents cells without treatment, while control (CTRL) indicates cells treated with 0.1% DMSO as vehicle. Cells were treated with ETO and Tph, and COMB for 24 h. Data represented as mean ± SD of 3 independent experiments with n=3 dishes/treatment group/experiment. (b) Effect of ETO, Tph and COMB treatments on COX2, carbonyl protein, SOD activity, nitrate/nitrite and nitrite in 5XFAD mice brains homogenate. Mice were treated with ETO, Tph or COMB at 10 mg/kg/day each for one month. Data are presented as mean ± SEM for n=5–6 mice in each group. ns = not significant; & p < 0.05 compared to CTRL; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CTRL; ## p < 0.01, ### p < 0.001represent COMB compared to ETO alone; @@ p < 0.01, @@@ p < 0.001represent COMB compared to Tph alone.
Figure 6.
Figure 6.
Combination (COMB) treatment significantly reduced astrogliosis marker GFAP. Representative brain sections of 5XFAD mice treated with etodolac (ETO), α-tocopherol (Tph), and COMB stained with anti-GFAP antibody (red) to detect activated astrocytes and 6E10 antibody (green) to detect total Aβ, and GFAP optical density quantitation in both brain regions, cortex and hippocampus. Scale bar, 50 μm. Mice were treated with ETO, Tph or COMB at 10 mg/kg/day each for 30 days. Data are presented as mean ± SEM for n=9 mice for control group and n=5 mice for treatment groups. ns = not significant; *** p < 0.001 compared to CTRL; ### p < 0.001represents COMB compared to ETO alone; @@@ p < 0.001represents COMB compared to Tph alone.
Figure 7.
Figure 7.
Combination (COMB) treatment significantly increased sAPPα production and synaptic markers PSD-95 and SNAP-25. Representative Western blot and densitometry analysis of (a) full length APP (fAPP), sAPPα and sAPPβ, and (b) PSD-95 and SNAP-25 in SH-SY5Y-APP cells treated with etodolac (ETO), α-tocopherol (Tph), and COMB 10μM each for 24 h. Data represented as mean ± SD of 3 independent experiments with n=3 dishes/treatment group/experiment. (c) Representative Western blot and densitometry analysis of fAPP, sAPPα and sAPPβ, and (d) PSD-95 and SNAP-25 expressions in the brain homogenates of 5XFAD mice. Mice were treated with ETO, Tph or COMB at 10 mg/kg/day each for 30 days. Data are presented as mean ± SEM for n=5–6 mice in each group. ns = not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CTRL; # p < 0.05, ## p < 0.01 represent COMB compared to ETO alone; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001represent COMB compared to Tph alone.
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