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. 2013 Jan;54(1):58-68.
doi: 10.1111/j.1600-079X.2012.01022.x. Epub 2012 Jul 23.

Overexpression of melatonin membrane receptors increases calcium-binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

Arabinda Das  1 , Gerald Wallace 4th, Russel J Reiter, Abhay K Varma, Swapan K Ray, Naren L Banik
Affiliations

Overexpression of melatonin membrane receptors increases calcium-binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

Arabinda Das et al. J Pineal Res. 2013 Jan.

Abstract

Melatonin has shown particular promise as a neuroprotective agent to prevent motoneuron death in animal models of both amyotrophic lateral sclerosis (ALS) and spinal cord injuries (SCI). However, an understanding of the roles of endogenous melatonin receptors including MT1, MT2, and orphan G-protein receptor 50 (GPR50) in neuroprotection is lacking. To address this deficiency, we utilized plasmids for transfection and overexpression of individual melatonin receptors in the ventral spinal cord 4.1 (VSC4.1) motoneuron cell line. Receptor-mediated cytoprotection following exposure to glutamate at a toxic level (25 μm) was determined by assessing cell viability, apoptosis, and intracellular free Ca(2+) levels. Our findings indicate a novel role for MT1 and MT2 for increasing expression of the calcium-binding proteins calbindin D28K and parvalbumin. Increased levels of calbindin D28K and parvalbumin in VSC4.1 cells overexpressing MT1 and MT2 were associated with cytoprotective effects including inhibition of proapoptotic signaling, downregulation of inflammatory factors, and expression of prosurvival markers. Interestingly, the neuroprotective effects conferred by overexpression of MT1 and/or MT2 were also associated with increases in the estrogen receptor β (ERβ): estrogen receptor α (ERα) ratio and upregulation of angiogenic factors. GPR50 did not exhibit cytoprotective effects. To further confirm the involvement of the melatonin receptors, we silenced both MT1 and MT2 in VSC4.1 cells using RNA interference technology. Knockdown of MT1 and MT2 led to an increase in glutamate toxicity, which was only partially reversed by melatonin treatment. Taken together, our findings suggest that the neuroprotection against glutamate toxicity exhibited by melatonin may depend on MT1 and MT2 but not GPR50.

Keywords: G‐protein receptor 50; apoptosis; calbindin D28K; calpain; glutamate toxicity; melatonin receptors; parvalbumin; ventral spinal cord 4.1.

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Figures

Fig. 1.
Fig. 1.
Melatonin receptor (MT1 and MT2) overexpression prevented ventral spinal cord 4.1 (VSC4.1) motoneuron death. Plasmid-mediated increase (P↑) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 μm L-glutamate (LGA) (24 h exposure), 100 nm melatonin (24 h pretreatment) + 25 μm LGA (24 h), MT1-overexpressed cells + 25 μm LGA (24 h), MT2-overexpressed cells + 25 μm LGA (24 h), MT1 + MT2–overexpressed cells + 25 μm LGA (24 h), GPR50-overexpressed cells + 25 μm LGA (24 h). (A) ApopTagassay showing representative images from each treatment group. Arrows indicate apoptotic cells. (B) Bar graphs indicating the percentage of apoptotic cells (ApopTag assay) and viability (trypan blue dye exclusion test) counted from each group. (C) Representative pictures show levels of MT1, MT2, GPR50, and β-actin levels (Western blotting). (D) Bar graphs indicating the changes in expression of MT1, MT2, and GPR50 over Con.
Fig. 2.
Fig. 2.
Overexpression of MT1 and MT2 increases calbindin D28K and parvalbumin for suppressing Ca2+ rise and calpain:calpastatin ratio in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P↑) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 μm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 μm LGA (24 h exposure), MT1-overexpressed cells + 25 μm LGA (24 h exposure), MT2-overexpressed cells + 25 μm LGA (24 h exposure), MT1 + MT2–overexpressed cells + 25 μm LGA (24 h exposure), GPR50-overexpressed cells + 25 μm LGA (24 h exposure). (A) Determination of intracellular free Ca2+ levels at 24 h. (B) Western blot analysis to show levels of calbindin D28K, parvalbumin, calpain, calpastatin, and β-actin. (C) Bar graphs indicating the changes in expression of calbindin D28K and parvalbumin over Con. (D) Densitometric analysis showing the calpain:calpastatin ratio.
Fig. 3.
Fig. 3.
Overexpression of MT1 and MT2 increase estrogen receptor (ERβ:ERα) ratio and suppression inflammatory factors in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P↑) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 μm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 μm LGA (24 h), MT1-overexpressed cells + 25 μm LGA (24 h), MT2-overexpressed cells + 25 μm LGA (24 h), MT1 + MT2–overexpressed cells + 25 μm LGA (24 h), GPR50-overexpressed cells + 25 μm LGA (24 h). (A) Western blot analysis to show levels of ERβ, ERα, NF-κB, COX-2, and β-actin. (B) Densitometric analysis showing the ERβ: ERα ratio. (C) Bar graphs indicating the changes in the expression of NF-κB and COX-2 over Con.
Fig. 4
Fig. 4
Overexpression of MT1 and MT2 increases survival and angiogenesic factors in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P↑) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 μm L-glutamate (LGA) (24 h exposure), 100 nm melatonin (24 h pretreatment) + 25 μm LGA (24 h exposure), MT1-overexpressed cells + 25 μm LGA (24 h), MT2-overexpressed cells + 25 μm LGA (24 h exposure), MT1 + MT2–overexpressed cells + 25 μm LGA (24 h exposure), GPR50-overexpressed cells + 25 μm LGA (24 h exposure). (A) Western blot analysis to show levels of p-Akt, Bcl-2, p-Bad, Flk-1, Flt-1, VEGF, and β-actin. (B) Bar graphs indicating the changes in expression of p-Akt, Bcl-2, and p-Bad over Con. (C) Bar graphs indicating changes in the expression of Flk-1, Flt-1, and VEGF over Con.
Fig. 5.
Fig. 5.
Overexpression of MT1 and MT2 suppresses apoptotic pathways in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P↑) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 μm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 μm LGA (24 h), MT1-overexpressed cells + 25 μm LGA (24 h), MT2-overexpressed cells + 25 μm LGA (24 h), MT1 + MT2–overexpressed cells + 25 μm LGA (24 h), GPR50-overexpressed cells + 25 μm LGA (24 h). (A) Western blot analysis to show levels of Bax, Bcl-2, active caspase-9, active caspase-3, and β-actin. (B) Densitometric analysis showing the Bax:Bcl-2 ratio. (C) Bar graphs indicating the changes in expression of active caspase-9 and active caspase-3 over Con. (D) Colorimetric determination of caspase-9 and caspase-3 activities.
Fig. 6.
Fig. 6.
Silencing MT1 and MT2 enhanced glutamate toxicity in ventral spinal cord 4.1 (VSC4.1) cells. We indicated the use of RNA interference (RNAi) to cause a decrease in expression. Treatment groups: control cells (Con), 25 μm L-glutamate (LGA) (24 h exposure), MT1-silenced cells + 25 μm LGA (24 h exposure); MT2-silenced cells + 25 μm LGA (24 h), MT1 + MT2-silenced cells + 25 μm LGA (24 h exposure); G-protein receptor 50 (GPR50) silenced cells + 25 μm LGA (24 h), 25 μm LGA (24 h), 25 μm LGA + 100 nm melatonin (24 h exposure), MT1-silenced cells + 25 μm LGA+ 100 nm melatonin (24h exposure), MT2 silenced cells + 25 μm LGA + 100 nm melatonin (24 h exposure), MT1 + MT2-silenced cells + 25 μm LGA + 100 nm melatonin (24 h exposure), GPR50 silenced cells + 25 μm LGA + 100 nm melatonin (24 h exposure). (A) Representative pictures showing levels of MT1, MT2, GPR50, and β-actin levels (Western blotting). (B) Bar graphs indicating the percentage of viability (trypan blue dye exclusion test) counted from each group. (C) Colorimetric determination of caspase-3 activity.
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