The inhibition of apoptosis by melatonin in VSC4.1 motoneurons exposed to oxidative stress, glutamate excitotoxicity, or TNF-alpha toxicity involves membrane melatonin receptors

Abstract

Loss of motoneurons may underlie some of the deficits in motor function associated with the central nervous system (CNS) injuries and diseases. We tested whether melatonin, a potent antioxidant and free radical scavenger, would prevent motoneuron apoptosis following exposure to toxins and whether this neuroprotection is mediated by melatonin receptors. Exposure of VSC4.1 motoneurons to either 50 microm H(2)O(2), 25 microm glutamate (LGA), or 50 ng/mL tumor necrosis factor-alpha (TNF-alpha) for 24 h caused significant increases in apoptosis, as determined by Wright staining and ApopTag assay. Analyses of mRNA and proteins showed increased expression and activities of stress kinases and cysteine proteases and loss of mitochondrial membrane potential during apoptosis. These insults also caused increases in intracellular free [Ca(2+)] and activities of calpain and caspases. Cells exposed to stress stimuli for 15 min were then treated with 200 nm melatonin. Post-treatment of cells with melatonin attenuated production of reactive oxygen species (ROS) and phosphorylation of p38, MAPK, and JNK1, prevented cell death, and maintained whole-cell membrane potential, indicating functional neuroprotection. Melatonin receptors (MT1 and MT2) were upregulated following treatment with melatonin. To confirm the involvement of MT1 and MT2 in providing neuroprotection, cells were post-treated (20 min) with 10 microm luzindole (melatonin receptor antagonist). Luzindole significantly attenuated melatonin-induced neuroprotection, suggesting that melatonin worked, at least in part, via its receptors to prevent VSC4.1 motoneuron apoptosis. Results suggest that neuroprotection rendered by melatonin to motoneurons is receptor mediated and melatonin may be an effective neuroprotective agent to attenuate motoneuron death in CNS injuries and diseases.

Fig. 1

Post-treatment with melatonin prevented cell death and preserved functionality. (A) Post-treatment with melatonin prevented H₂O₂, LGA, or TNF-α mediated decrease in VSC4.1 cell viability. The trypan blue dye exclusion assay was used to assess cell viability in VSC4.1 cells. (B) Measurement of whole-cell membrane potential in VSC4.1 cells following treatments. Twelve treatment groups: control (CTL); 150 nM melatonin (24 h); 10 μM luzindole (24 h); 50 μM H₂O₂ (24 h); 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin; 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 25 μM LGA (24 h); 25 μM LGA (24 h) + post-treatment (15 min) of melatonin; 25 μM LGA (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin; 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole.

Fig. 2

Post-treatment with melatonin prevented apoptotic death of VSC4.1 cells exposed to H₂O₂, LGA, or TNF-α. (A) Photomicrographs showing representative cells from each treatment group followingg Wright staining. The arrows indicate apoptotic cells. (B) Photomicrographs showing representative cells from each treatment group following ApopTag assay. (C) Bar graphs indicating the percentage of apoptotic cells in each group (based on Wright staining). Twelve treatment groups: control (CTL); 150 nM melatonin (24 h); 10 μM luzindole (24 h); 50 μM H₂O₂ (24 h); 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin; 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 25 μM LGA (24 h); 25 μM LGA (24 h) + post-treatment (15 min) of melatonin; 25 μM LGA (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin; 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole.

Fig. 3

RT-PCR and Western blotting for determing expression of melatonin receptors in VSC4.1 cells. (A) Representative pictures to show levels of melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2), and GAPDH at mRNA levels (RT-PCR). (B) Bar graphs to indicate the percentage of change in MT1 and MT2 expression relative to control (CTL) at mRNA level. (C) Representative pictures to show levels of MT1 and MT2, and β-Actin at protein levels (Western blotting). Twelve treatment groups: control (CTL); 150 nM melatonin (24 h); 10 μM luzindole (24 h); 50 μM H₂O₂ (24 h); 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin; 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 25 μM LGA (24 h); 25 μM LGA (24 h) + post-treatment (15 min) of melatonin; 25 μM LGA (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin; 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole.

Fig. 4

Determination of ROS production, p38 MAPK and JNK1 phosphorylation in VSC4.1 cells. (A) Inhibition of ROS production. Treatments (0, 30, 60, 90, 120, 150, 180, and 1440 min) in the presence of 5 μM 2,7-dichlorofluorescin diacetate (DCF-DA). (B) Western blotting to show levels of p-p38 MAPK, p38 MAPK, p-JNK-1 and β-actin. (C) Densitometric analysis showing percent change in optical density of the p-p38 MAPK and p-JNK-1 bands. Twelve treatment groups d: control (CTL); 150 nM melatonin (24 h); 10 μM luzindole (24 h); 50 μM H₂O₂ (24 h); 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin; 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 25 μM LGA (24 h); 25 μM LGA (24 h) + post-treatment (15 min) of melatonin; 25 μM LGA (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin; 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole.

Fig. 5

Determination of caspase-8 activation and Bid cleavage. (A) Western blotting to show levels of caspase-8, β-actin, tBid, and COX4. (B) Densitometric analysis to show percent change in optical density of the caspase-8 and tBid bands. (C) Colorimetric determination of caspase-8 activity. Twelve treatment groups: control (CTL); 150 nM melatonin (24 h); 10 μM luzindole (24 h); 50 μM H₂O₂ (24 h); 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin; 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 25 μM LGA (24 h); 25 μM LGA (24 h) + post-treatment (15 min) of melatonin; 25 μM LGA (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin; 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole.

Fig. 6

Examination of components involved in mitochondrial pathway of apoptosis in VSC4.1 cells. Alterations in Bax and Bcl-2 expression at mRNA and protein levels. Representative pictures to show Bax, Bcl-2, and GAPDH at (A) mRNA levels (RT-PCR) and (B) protein levels (Western blotting). (C) Densitometric analysis show the Bax:Bcl-2 ratio. (D) JC-1 ratio (590 nm/530 nm) in cells after the treatments for different times (30, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, and 1440 min). (E) Western blotting for cytochrome c, COX4, caspase-9, and β-actin. (F) Densitometric analysis to show percent change in optical density of the mitochondrial and cytosolic 15 kD cytochrome c and 39 kD active caspase-9. (G) Determination of caspase-9 activity using a colorimetric assay. Twelve treatment groups: control (CTL); 150 nM melatonin (24 h); 10 μM luzindole (24 h); 50 μM H₂O₂ (24 h); 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin; 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 25 μM LGA (24 h); 25 μM LGA (24 h) + post-treatment (15 min) of melatonin; 25 μM LGA (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin; 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole.

Fig. 7

Examination of increase in intracellular free [Ca²⁺], activation of calpain and caspase-3, and CAD. (A) Determination of intracellular free [Ca²⁺]. (B) Western blotting to show levels of calpain, spectrin breakdown product (SBDP), and active caspase-3. (C) Densitometric analysis to document percent change in optical density of 80 kD calpain, 145 kD SBDP, 120 kD SBDP, and 20 kD caspase-3. (C) Determination of caspase-3 activity by a colorimetric assay. (E) Western blotting to show levels of ICAD, CAD, and β-actin. (F) Densitometric analysis to show percent change in optical density of the 45 kD ICAD, and 40 kD CAD. Twelve treatment groups:control (CTL); 150 nM melatonin (24 h); 10 μM luzindole (24 h); 50 μM H₂O₂ (24 h); 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin; 50 μM H₂O₂ (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 25 μM LGA (24 h); 25 μM LGA (24 h) + post-treatment (15 min) of melatonin; 25 μM LGA (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole; 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin; 50 ng/ml TNF-α (24 h) + (15 min post-treat) melatonin + (20 min post-treat) luzindole. Das et al. (JPI-OM-10-09-0154)