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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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Glutathione peroxidase (GPx) levels in Mn-treated astrocytes.This is a representative histogram of fold-changes in GPxlevels by densitometry, using Western blot analysis. There was a trend for elevated levels of GPx levels after 6 hrs of 500 µM of Mn exposure. The fold-changes observed for GPx-1 were not significant. Similarly, OTC alone or OTC pretreatment followed by Mn exposure did not lead to any changes in GPx-1 protein levels. These data were collected from N = 5 replicates similar to the biological samples used to measure SOD-2 fold changes. Results were independently normalized to β-actin and to control prior to statistical analysis.
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pone-0094753-g003: Glutathione peroxidase (GPx) levels in Mn-treated astrocytes.This is a representative histogram of fold-changes in GPxlevels by densitometry, using Western blot analysis. There was a trend for elevated levels of GPx levels after 6 hrs of 500 µM of Mn exposure. The fold-changes observed for GPx-1 were not significant. Similarly, OTC alone or OTC pretreatment followed by Mn exposure did not lead to any changes in GPx-1 protein levels. These data were collected from N = 5 replicates similar to the biological samples used to measure SOD-2 fold changes. Results were independently normalized to β-actin and to control prior to statistical analysis.

Mentions: We subsequently tested whether the changes in F2-IsoP levels in Mn exposed astrocytes correlated with oxidative stress response. We measured protein levels of SOD-2 and GPx-1. We found that levels of SOD-2 were elevated after 6 hrs of Mn exposure (Fig. 2). SOD-2 protein levels increased 1.73±0.06 fold after Mn exposure (Fig. 2). Pre-treatment of these cells with OTC (for 24 hrs) prior to Mn exposure (for 6 hrs) led to further elevation of SOD-2 (1.8±0.13). Interestingly, OTC alone also increased SOD-2 levels by 1.4±0.15 fold (Fig. 2). On the other hand, Mn exposure increased GPx-1 levels by 1.3±0.2 fold, compared to the controls with no statistical significance (Fig. 3). OTC pre-treatment of these astrocytes in the presence or absence of Mn did not lead to changes in GPx1 levels (Fig 3).


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)

Glutathione peroxidase (GPx) levels in Mn-treated astrocytes.This is a representative histogram of fold-changes in GPxlevels by densitometry, using Western blot analysis. There was a trend for elevated levels of GPx levels after 6 hrs of 500 µM of Mn exposure. The fold-changes observed for GPx-1 were not significant. Similarly, OTC alone or OTC pretreatment followed by Mn exposure did not lead to any changes in GPx-1 protein levels. These data were collected from N = 5 replicates similar to the biological samples used to measure SOD-2 fold changes. Results were independently normalized to β-actin and to control prior to statistical analysis.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0094753-g003: Glutathione peroxidase (GPx) levels in Mn-treated astrocytes.This is a representative histogram of fold-changes in GPxlevels by densitometry, using Western blot analysis. There was a trend for elevated levels of GPx levels after 6 hrs of 500 µM of Mn exposure. The fold-changes observed for GPx-1 were not significant. Similarly, OTC alone or OTC pretreatment followed by Mn exposure did not lead to any changes in GPx-1 protein levels. These data were collected from N = 5 replicates similar to the biological samples used to measure SOD-2 fold changes. Results were independently normalized to β-actin and to control prior to statistical analysis.
Mentions: We subsequently tested whether the changes in F2-IsoP levels in Mn exposed astrocytes correlated with oxidative stress response. We measured protein levels of SOD-2 and GPx-1. We found that levels of SOD-2 were elevated after 6 hrs of Mn exposure (Fig. 2). SOD-2 protein levels increased 1.73±0.06 fold after Mn exposure (Fig. 2). Pre-treatment of these cells with OTC (for 24 hrs) prior to Mn exposure (for 6 hrs) led to further elevation of SOD-2 (1.8±0.13). Interestingly, OTC alone also increased SOD-2 levels by 1.4±0.15 fold (Fig. 2). On the other hand, Mn exposure increased GPx-1 levels by 1.3±0.2 fold, compared to the controls with no statistical significance (Fig. 3). OTC pre-treatment of these astrocytes in the presence or absence of Mn did not lead to changes in GPx1 levels (Fig 3).

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