J Clin Neurol.  2010 Sep;6(3):127-137. 10.3988/jcn.2010.6.3.127.

Melatonin Potentiates the Neuroprotective Properties of Resveratrol Against Beta-Amyloid-Induced Neurodegeneration by Modulating AMP-Activated Protein Kinase Pathways

• Kwon KJ
• Kim HJ
• Shin CY
• Han SH

• KMID: 1462848
• DOI: http://doi.org/10.3988/jcn.2010.6.3.127

Abstract

BACKGROUND AND PURPOSE
Recent studies have demonstrated that resveratrol (RSV) reduces the incidence of age-related macular degeneration, Alzheimer's disease (AD), and stroke, while melatonin (MEL) supplementation reduces the progression of the cognitive impairment in AD patients. The purpose of this investigation was to assess whether the co-administration of MEL and RSV exerts synergistic effects on their neuroprotective properties against beta-amyloid (Abeta)-induced neuronal death.
METHODS
The neuroprotective effects of co-treatment with MEL and RSV on Abeta1-42 -induced cell death, was measured by MTT reduction assay. Abeta1-42 caused an increase in intracellular levels of reactive oxygen species (ROS), as assessed by H2-DCF-DA dye, and a reduction of total glutathione (GSH) levels and mitochondrial membrane potential, as assessed using monochlorobimane and rhodamine 123 fluorescence, respectively. Western blotting was used to investigate the intracellular signaling mechanism involved in these synergic effects.
RESULTS
We treated a murine HT22 hippocampal cell line with MEL or RSV alone or with both simultaneously. MEL and RSV alone significantly attenuated ROS production, mitochondrial membrane-potential disruption and the neurotoxicity induced by Abeta1-42. They also restored the Abeta1-42-induced depletion of GSH, back to within its normal range and prevented the Abeta1-42-induced activation of glycogen synthase kinase 3beta (GSK3beta). However, co-treatment with MEL and RSV did not exert any significant synergistic effects on either the recovery of the Abeta1-42-induced depletion of GSH or on the inhibition of Abeta1-42-induced GSK3beta activation. Abeta1-42 treatment increased AMP-activated protein kinase (AMPK) activity, which is associated with subsequent neuronal death. We demonstrated that MEL and RSV treatment inhibited the phosphorylation of AMPK.
CONCLUSIONS
Together, our results suggest that co-administration of MEL and RSV acts as an ef-fective treatment for AD by attenuating Abeta1-42-induced oxidative stress and the AMPK-dependent pathway.

Keyword

melatonin; resveratrol; neuroprotection; reactive oxygen species; glycogen synthase kinase 3beta; AMP-activated protein kinase

MeSH Terms

Alzheimer Disease
AMP-Activated Protein Kinases
Blotting, Western
Cell Death
Cell Line
Fluorescence
Glutathione
Glycogen Synthase Kinase 3
Glycogen Synthase Kinases
Humans
Incidence
Macular Degeneration
Melatonin
Membrane Potential, Mitochondrial
Neurons
Neuroprotective Agents
Oxidative Stress
Phosphorylation
Pyrazoles
Reactive Oxygen Species
Reference Values
Rhodamine 123
Stilbenes
Stroke
AMP-Activated Protein Kinases
Glutathione
Glycogen Synthase Kinase 3
Glycogen Synthase Kinases
Melatonin
Neuroprotective Agents
Pyrazoles
Reactive Oxygen Species
Rhodamine 123
Stilbenes

Figure

• 

  Fig. 1 Protective effects of MEL and RSV on Aβ1-42-induced cytotoxicity in HT22 hippocampal neuronal cells. A and B: HT22 cells were treated with the indicated concentrations of MEL (1, 10, 50, 100, and 500 µM) and RSV (0.1, 1, 10, and 20 µM) in the absence or presence of Aβ1-42 for 24h at 37℃. The viability of HT22 cells was determined by the MTT reduction assay after treatment with MEL, RSV, and Aβ1-42. MEL was administered 1-hr before Aβ1-42 treatment, while RSV was added to the medium along with Aβ1-42. C: Cotreatment effects of MEL (1 and 10 µM) and RSV (0.1 and 1 µM). Data are expressed as the mean±S.E.M values of three experiments. The data shown are representative of five independent experiments that gave similar results. **p<0.01 vs. control, †p<0.05, ‡p<0.01 vs. Aβ1-42 alone. MEL: melatonin, RSV: resveratrol, Aβ: β-amyloid.

• 

  Fig. 2 Antioxidative properties of MEL and RSV on Aβ1-42-induced oxidative stress in HT22 hippocampal neuronal cells. MEL was administered 1-h before Aβ1-42 treatment, while RSV was added to the medium along with the Aβ1-42. A: Effects of MEL and RSV on Aβ1-42-induced increase in DCF fluorescence. RSV at the indicated concentration was applied concomitantly with 1 µM Aβ1-42 for 6-h in HT22 cells. Intracellular ROS was evaluated by measuring H2DCF-DA fluorescence (490/530 nm), as described in the Materials and Methods. B: Effects of MEL and RSV on Aβ1-42-induced depletion of GSH. After 12-h of Aβ1-42 treatment, mBCl was applied to the medium and total GSH levels were determined spectrophotometrically at 390/480 nm. C: Effects of MEL and RSV on Δψm, as assessed by fluorescence microscopy using the fluorescence dye rhodamine 123, as described in the Materials and Methods. D: Measurement of Δψm by fluorospectrophotometry: MEL (100 µM), RSV (20 µM), M10R1 (MEL 10 µM+RSV 1 µM), and Tro (Trolox, 100 µM). Data are expressed as mean±S.E.M values. The data shown are representative of five independent experiments that gave similar results. *p<0.05, **p<0.01 vs. control, †p<0.05, ‡p<0.01 vs. Aβ1-42 alone. MEL: melatonin, RSV: resveratrol, Aβ: β-amyloid, DCF: dichlorofluorescein.

• 

  Fig. 3 Effects of MEL and RSV on Aβ1-42-induced MAPK signaling. A: HT22 cells were incubated with 1-µM Aβ1-42 for 30-min in the presence or absence of MEL and RSV at the indicated concentrations, and then harvested for Western blot analysis. Immunoblots of lysates from treated HT22 cells were probed with phospho-ERK (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), and phospho-JNK (Thr183/Tyr185) antibodies. As a loading control, total ERK/p38/JNK levels were also measured (lower panel). MEL and RSV attenuated Aβ1-42-induced activation of ERK and p38 MAPK. B: HT22 cells were treated with the indicated concentrations of PD (inhibitor of ERK), SB (inhibitor of p38 MAPK), and SP (inhibitor of JNK) in the presence of Aβ1-42 for 24-h at 37℃. The viability of HT22 cells was determined by the MTT reduction assay after treatment with PD, SB, and SP with Aβ1-42. Treatment with these MAPK inhibitors blocked the Aβ1-42-induced activation of ERK/p38 MAPK. Data are expressed as the mean±S.E.M values of four independent experiments. **p<0.01 vs. control, †p<0.05, ‡p<0.01 vs. Aβ1-42 alone. MEL: melatonin, RSV: resveratrol, Aβ: β-amyloid, MAPK: mitogen-activated protein kinase, ERK: extracellular signal related kinase.

• 

  Fig. 4 Inhibitory effects of MEL and RSV on Aβ1-42-induced GSK-3β activation. A: HT22 cells were incubated with 2 µM Aβ1-42 for 6-h in the presence or absence of the indicated concentrations of MEL and RSV, and harvested for Western blot analysis. Immunoblots of lysates from treated HT22 cells were probed with phospho-GSK3β (Ser9) and GSK3β antibodies. Actin levels were measured for the confirmation of equal amount of protein loading (lower panel). MEL and RSV inhibited the Aβ1-42-induced activation of GSK-3β. B: HT22 cells were treated with indicated concentrations of LiCl (inhibitor of GSK3β) in the presence of Aβ1-42 for 24-h at 37℃. The viability of HT22 cells was determined by the MTT reduction assay after treatment of LiCl with Aβ1-42. Data are expressed as the mean±S.E.M values of four independent experiments. **p<0.01 vs. control, †p<0.05, ‡p<0.01 vs. Aβ1-42 alone; RSV (20 µM), MEL (100 µM), MR (MEL 10 µM+RSV 1 µM), Li (LiCl, 1mM). MEL: melatonin, RSV: resveratrol, Aβ: β-amyloid.

• 

  Fig. 5 Effects of MEL and RSV on the activation of AMPK signaling. A: HT22 cells were incubated with 2 µM Aβ1-42 for 24-h in the presence or absence of the indicated concentrations of MEL and RSV, and then harvested for Western blot analysis. Immunoblots of lysates from treated HT22 cells were probed with phospho-AMPK (Thr172) and total AMPK antibodies. Actin levels were also measured to confirm equal amounts of protein loading (lower panel). MEL and RSV inhibited the Aβ1-42-induced activation of AMPK. B: HT22 cells were treated with Ara-A (inhibitor of AMPK) indicated concentrations, and Aβ1-42, or (C) the cells were treated with AICAR (activator of AMPK) without Aβ1-42 for 24-h at 37℃ for the MTT assay. The viability of HT22 cells was determined by the MTT reduction assay. The inhibitor of AMPK blocked Aβ1-42-induced neuronal cell death and the activator of AMPK augmented Aβ1-42-induced neuronal cell death. Data are expressed as the mean±S.E.M values of three independent experiments. **p<0.01 vs. control, †p<0.05, ‡p<0.01 vs. Aβ1-42 alone, §p<0.01 vs. drug only; RSV (20 µM), MEL (100µM), MR (MEL 10 µM+RSV 1 µM), AIC (AICAR, 10-100 µM), Ara-A (10-100 µM). MEL: melatonin, RSV: resveratrol, Aβ: β-amyloid, ERK: extracellular signal related kinase, AMPK: AMP-activated protein kinase.

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