The inhibition of apoptosis by melatonin in VSC4.1 motoneurons exposed to oxidative stress, glutamate excitotoxicity, or TNF-alpha toxicity involves membrane melatonin receptors.
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.
Das
Arabinda
A
Department of Neurosciences (Division of Neurology), Medical University of South Carolina, Charleston, SC 29425, USA.
McDowell
Misty
M
Pava
Matthew J
MJ
Smith
Joshua A
JA
Reiter
Russel J
RJ
Woodward
John J
JJ
Varma
Abhay K
AK
Ray
Swapan K
SK
Banik
Naren L
NL
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000633:toxicity*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008550:Melatonin / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D009046:Motor Neurons / Q000187:drug effects* / Q000378:metabolism*; D018384:Oxidative Stress; D044122:Receptor, Melatonin, MT1 / Q000378:metabolism; D044123:Receptor, Melatonin, MT2 / Q000378:metabolism; D044094:Receptors, Melatonin / Q000502:physiology*; D014363:Tryptamines / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*
D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; D044122:Receptor, Melatonin, MT1; D044123:Receptor, Melatonin, MT2; D044094:Receptors, Melatonin; D014363:Tryptamines; D014409:Tumor Necrosis Factor-alpha; C057154:luzindole; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; D053181:Caspase 8; D008550:Melatonin; D002118:Calcium