Reduction of Mitophagy-Related Oxidative Stress and Preservation of Mitochondria Function Using Melatonin Therapy in an HT22 Hippocampal Neuronal Cell Model of Glutamate-Induced Excitotoxicity

Abstract

Recent evidence indicates that autophagy-mediated mitochondrial homeostasis is crucial for oxidative stress-related brain damage and repair. The highest concentration of melatonin is in the mitochondria of cells, and melatonin exhibits well-known antioxidant properties. We investigated the impact and mechanism involved in mitochondrial function and the mitochondrial oxidative stress/autophagy regulator parameters of glutamate cytotoxicity in mouse HT22 hippocampal neurons. We tested the hypothesis that melatonin confers neuroprotective effects via protecting against mitochondrial impairment and mitophagy. Cells were divided into four groups: the control group, melatonin alone group, glutamate injury group, and melatonin pretreatment group. We found that glutamate induced significant changes in mitochondrial function/oxidative stress-related parameters. Leptin administration preserved mitochondrial function, and this effect was associated with increased superoxide dismutase, glutathione (GSH), and mitochondrial membrane potential and decreased GSSG (oxidized glutathione) and mitochondrial reactive oxygen species. Melatonin significantly reduced the fluorescence intensity of mitophagy via the Beclin-1/Bcl-2 pathway, which involves Beclin-1 and Bcl-2 proteins. The mitophagy inhibitor CsA corrected these glutamate-induce changes, as measured by the fluorescence intensity of Mitophagy-Tracker Red CMXROS, mitochondrial ROS, and mitochondrial membrane potential changes. These findings indicate that melatonin exerts neuroprotective effects against glutamate-induced excitotoxicity by reducing mitophagy-related oxidative stress and maintaining mitochondrial function.

Keywords: HT22; glutamate; melatonin; mitochondria; mitophagy.

Figure 1

Observation of cell morphology in each group under light microscopy (×10).

Figure 2

Protective effect of melatonin on glutamate-induced injury in HT22 cells. (A) Effect of melatonin on cell survival rate: F = 27.242, P < 0.001; control group vs. glutamate injury group: P < 0.001, glutamate injury group vs. melatonin pretreatment group: P = 0.005; (B) Effect of melatonin on the release of LDH: F = 11.553, P < 0.001; control group vs. glutamate injury group: P = 0.011, glutamate injury group vs. melatonin pretreatment group: P = 0.039; *P < 0.05, **P < 0.01. ns, not significant (n = 6/group).

Figure 3

Effect of melatonin on glutamate-induced oxidation of HT22 cells. (A) Effect of melatonin on the activity of SOD in cells: F = 19.937, P < 0.001; control group vs. glutamate injury group: P < 0.001, glutamate injury group vs. melatonin pretreatment group: P = 0.034; (B) Effect of melatonin on the intracellular GSH concentration: F = 9.315, P < 0.001; control group vs. glutamate injury group: P = 0.011, glutamate injury group vs. melatonin pretreatment group: P = 0.044; (C) Effect of melatonin on the intracellular GSSG concentration: F = 5.921, P = 0.005; control group vs. glutamate injury group: P = 0.014, glutamate injury group vs. melatonin pretreatment group: P = 0.045; (D) Effects of melatonin on GSH/GSSG ratio in cells: F = 8.363, P = 0.001; control group vs. glutamate injury group: P = 0.011, glutamate injury group vs. melatonin pretreatment group: P = 0.039; *P < 0.05, **P < 0.01. ns, not significant (n = 6/group).

Figure 4

Effects of melatonin on glutamate-induced mitochondrial membrane potential changes by Mito-Tracker. (A) Detection of cell mitochondrial membrane potential changes by flow cytometry; (B) Fluorescence intensity of Mito-Tracker Red CMXROS in cells; (C) Quantitative analysis of mean fluorescence intensity of Mito-Tracker Red CMXROS in cells: F = 10.946, P < 0.001; control group vs. glutamate injury group: P = 0.011, glutamate injury group vs. melatonin pretreatment group: P = 0.035; *P < 0.05. ns, not significant (n = 6/group).

Figure 5

Effect of melatonin on glutamate-induced mitochondrial membrane potential changes using JC-1.

Figure 6

Effect of melatonin on mitochondrial reactive oxygen species (ROS) accumulation in cells. (A) Detection of ROS content in mitochondria using flow cytometry; (B) Fluorescence intensity of ROS in mitochondria; (C) Quantitative analysis of average fluorescence intensity of ROS in mitochondria: F = 24.903, P < 0.001; control group vs. glutamate injury group: P < 0.001, glutamate injury group vs. melatonin pretreatment group: P < 0.001; **P < 0.01. ns, not significant (n = 6/group).

Figure 7

Effect of melatonin on glutamate-induced mitophagy in HT22 cells. (A) Fluorescence intensity of mitochondrial Mtphagy Dye and Lyso Dye in cells; (B) Quantitative analyses of average fluorescence intensity of mitochondrial Mtphagy Dye in cells: F = 26.214, P < 0.001; control group vs. glutamate injury group: P < 0.001, glutamate injury group vs. melatonin pretreatment group: P < 0.001; **P < 0.01. ns, not significant (n = 6/group).

Figure 8

Effect of melatonin on the glutamate-induced expression of mitochondrial autophagy. Bcl-2/actin: F = 5.888, P = 0.005; control group vs. glutamate injury group: P = 0.010, glutamate injury group vs. melatonin pretreatment group: P = 0.013; Beclin-1/actin: F = 6.509, P = 0.003; control group vs. glutamate injury group: P = 0.011, glutamate injury group vs. melatonin pretreatment group: P = 0.044; Beclin-1/Bcl-2: F = 6.625, P = 0.003; control group vs. glutamate injury group: P = 0.010, glutamate injury group vs. melatonin pretreatment group: P = 0.029; *P < 0.05 (n = 6/group).

Figure 9

Effect of cyclosporine A (CsA) on glutamate-induced mitochondrial autophagy. (A) Fluorescence intensity of Mito-Tracker Red CMXROS in cells; (B) Quantitative analyses of mean fluorescence intensity of Mito-Tracker Red CMXROS in cells: t = 3.074, P = 0.012; (C) Mitochondrial membrane potential was detected using flow cytometry and Mito-Tracker; (D) Fluorescence intensity of mitochondrial ROS; (E) Quantitative analysis of mean fluorescence intensity of mitochondrial ROS: t = 4.511, P = 0.001; (F) Detection of ROS content in cell mitochondria using flow cytometry; (G) Fluorescence intensity of mitochondrial Mtphagy Dye and Lyso Dye in cells; (H) Quantitative analyses of mean fluorescence intensity of mitochondrial Mtphagy Dye in cells: t = 5.687, P < 0.001; (I) Expression of mitochondrial autophagy-related proteins in cells: Bcl-2/actin: t = 2.799, P = 0.019; Beclin-1/actin: t = 2.856, P = 0.017; Beclin-1/Bcl-2: t = 4.218, P = 0.002; (J) Observation of mitochondrial membrane potential changes using JC-1. *P < 0.05, **P < 0.01 (n = 6/group).