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Activation of MAPK and FoxO by manganese (Mn) in rat neonatal primary astrocyte cultures.

Exil V, Ping L, Yu Y, Chakraborty S, Caito SW, Wells KS, Karki P, Lee E, Aschner M - PLoS ONE (2014)

Bottom Line: Pre-treatment of cultured cells with (R)-(-)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO.At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged.FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Thomas P. Graham Division of Pediatric Cardiology, Monroe Carrell Jr. Children's Hospital, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

ABSTRACT
Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinson's disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl2 led to increased levels of FoxO (dephosphorylated and phosphorylated) compared with control cells (P<0.01). p-FoxO disappeared from the cytosol upon Mn exposure. Pre-treatment of cultured cells with (R)-(-)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events compensating for Mn-induced neurotoxicity and may serve as valuable targets for neuroprotection in the setting of Mn-induced neurodegeneration.

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OTC attenuates Mn-induced increased F2-Isoprostane levels in astrocytes.The histogram represents changes in values for Isoprostane levels in pg/mg of protein with Mn treatment in the presence or absence of OTC. F2-IsoP levels were significantly elevated after 6 hrs of 500 µM of Mn exposure alone. Pre-treatment with 1 mM of OTC for 24 hrs markedly reduced the effect of high doses of Mn. OTC alone did not lead to significant changes in F2-IsoP levels in astrocytes. Data was collected from N = 4 replicates. Isoprostane levels are expressed as Mean ± SD, * p<0.05.
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pone-0094753-g001: OTC attenuates Mn-induced increased F2-Isoprostane levels in astrocytes.The histogram represents changes in values for Isoprostane levels in pg/mg of protein with Mn treatment in the presence or absence of OTC. F2-IsoP levels were significantly elevated after 6 hrs of 500 µM of Mn exposure alone. Pre-treatment with 1 mM of OTC for 24 hrs markedly reduced the effect of high doses of Mn. OTC alone did not lead to significant changes in F2-IsoP levels in astrocytes. Data was collected from N = 4 replicates. Isoprostane levels are expressed as Mean ± SD, * p<0.05.

Mentions: First, we assessed the stress signals underlying Mn exposure and oxidative stress response by measuring astrocytic F2-IsoP levels. We found that F2-IsoP levels increased in response to Mn exposure. IsoP levels were 171.5±23.19 pg/mg protein for control cells, compared with 269.9±23.19 pg/mg protein after 6 hrs in Mn exposed astrocytes (Fig.1). F2-IsoP levels in astrocytes pre-treated with OTC followed by Mn exposure for 6 hrs were statistically indistinguishable from controls. OTC treatment alone did not cause changes in F2-IsoP levels. F2-IsoP levels in OTC pre-treated astrocytes for 24 hr followed by 6 hours of MnCl2 exposure were 141.7±15.02 pg/mg protein. In astrocytes treated with OTC alone F2IsoP levels were 187.9±32.93 pg/mg protein (Fig. 1).


Activation of MAPK and FoxO by manganese (Mn) in rat neonatal primary astrocyte cultures.

Exil V, Ping L, Yu Y, Chakraborty S, Caito SW, Wells KS, Karki P, Lee E, Aschner M - PLoS ONE (2014)

OTC attenuates Mn-induced increased F2-Isoprostane levels in astrocytes.The histogram represents changes in values for Isoprostane levels in pg/mg of protein with Mn treatment in the presence or absence of OTC. F2-IsoP levels were significantly elevated after 6 hrs of 500 µM of Mn exposure alone. Pre-treatment with 1 mM of OTC for 24 hrs markedly reduced the effect of high doses of Mn. OTC alone did not lead to significant changes in F2-IsoP levels in astrocytes. Data was collected from N = 4 replicates. Isoprostane levels are expressed as Mean ± SD, * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008430&req=5

pone-0094753-g001: OTC attenuates Mn-induced increased F2-Isoprostane levels in astrocytes.The histogram represents changes in values for Isoprostane levels in pg/mg of protein with Mn treatment in the presence or absence of OTC. F2-IsoP levels were significantly elevated after 6 hrs of 500 µM of Mn exposure alone. Pre-treatment with 1 mM of OTC for 24 hrs markedly reduced the effect of high doses of Mn. OTC alone did not lead to significant changes in F2-IsoP levels in astrocytes. Data was collected from N = 4 replicates. Isoprostane levels are expressed as Mean ± SD, * p<0.05.
Mentions: First, we assessed the stress signals underlying Mn exposure and oxidative stress response by measuring astrocytic F2-IsoP levels. We found that F2-IsoP levels increased in response to Mn exposure. IsoP levels were 171.5±23.19 pg/mg protein for control cells, compared with 269.9±23.19 pg/mg protein after 6 hrs in Mn exposed astrocytes (Fig.1). F2-IsoP levels in astrocytes pre-treated with OTC followed by Mn exposure for 6 hrs were statistically indistinguishable from controls. OTC treatment alone did not cause changes in F2-IsoP levels. F2-IsoP levels in OTC pre-treated astrocytes for 24 hr followed by 6 hours of MnCl2 exposure were 141.7±15.02 pg/mg protein. In astrocytes treated with OTC alone F2IsoP levels were 187.9±32.93 pg/mg protein (Fig. 1).

Bottom Line: Pre-treatment of cultured cells with (R)-(-)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO.At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged.FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Thomas P. Graham Division of Pediatric Cardiology, Monroe Carrell Jr. Children's Hospital, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

ABSTRACT
Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinson's disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl2 led to increased levels of FoxO (dephosphorylated and phosphorylated) compared with control cells (P<0.01). p-FoxO disappeared from the cytosol upon Mn exposure. Pre-treatment of cultured cells with (R)-(-)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events compensating for Mn-induced neurotoxicity and may serve as valuable targets for neuroprotection in the setting of Mn-induced neurodegeneration.

Show MeSH
Related in: MedlinePlus