Activation of MAPK and FoxO by manganese (Mn) in rat neonatal primary astrocyte cultures.
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.
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.
Vernat
V
Exil
Department of Pediatrics, Division of Pediatric Toxicology, Monroe Carrell Jr. Children's Hospital, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
Li
L
Ping
Department of Pediatrics, Division of Pediatric Toxicology, Monroe Carrell Jr. Children's Hospital, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
Yingchun
Y
Yu
Department of Pediatrics, Division of Pediatric Toxicology, Monroe Carrell Jr. Children's Hospital, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
Sudipta
S
Chakraborty
Department of Pediatrics, Division of Pediatric Toxicology, Monroe Carrell Jr. Children's Hospital, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
Samuel W
SW
Caito
Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
K Sam
KS
Wells
Department of Physiology, Meharry Medical College, Nashville, Tennessee, United States of America.
Pratap
P
Karki
Department of Physiology, Meharry Medical College, Nashville, Tennessee, United States of America.
Eunsook
E
Lee
Department of Pediatrics, Division of Pediatric Toxicology, Monroe Carrell Jr. Children's Hospital, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
Michael
M
Aschner
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D008345:Manganese / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013482:Superoxide Dismutase / Q000096:biosynthesis; D053778:Thiazolidines / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D000099156:Superoxide Dismutase 2
D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D008345:Manganese; D020928:Mitogen-Activated Protein Kinases; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D051057:Proto-Oncogene Proteins c-akt; D011761:Pyrrolidonecarboxylic Acid; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; D053778:Thiazolidines; D014157:Transcription Factors; D000099156:Superoxide Dismutase 2; C499515:FOXO3 protein, rat; C501429:Ppargc1a protein, rat; C029536:2-oxothiazolidine-4-carboxylic acid