Glutathione production is regulated via distinct pathways in stressed and non-stressed cortical neurons

Abstract

Peroxynitrite-mediated damage has been linked to numerous neurological and neurodegenerative diseases, including stroke, Alzheimer's and Parkinson's Diseases, amyotrophic lateral sclerosis and multiple sclerosis. Studies on the toxic effects of peroxynitrite in neurons have focused primarily on adverse effects resulting from the nitration of cellular proteins as the principal mode of toxicity while the consequences of the modulation of kinase pathways by peroxynitrite have received relatively less attention. Our results show that treatment of primary rat neurons with the peroxynitrite donor, SIN-1, leads to decreases in glutathione (GSH) levels and cell viability via a novel extracellular-signal-related kinase (ERK)/c-Myc phosphorylation pathway and a reduction in the nuclear expression of NF-E2-related factor-2 (Nrf2) that down-regulate the expression of glutamate cysteine ligase, the rate limiting enzyme for GSH synthesis. The flavonoid fisetin protects against the SIN-1-mediated alterations in ERK/c-Myc phosphorylation, nuclear Nrf2 levels, glutamate cysteine ligase levels, GSH concentration and cell viability. We also show that inhibition of mitogen-activated protein kinase kinase or Raf kinase can increase GSH levels in unstressed primary rat neurons through the same ERK/c-Myc phosphorylation pathway. Together, these results demonstrate that distinct signaling pathways modulate GSH metabolism in unstressed and stressed cortical neurons.

Figure 1

Effects of SIN-1, peroxynitrite and fisetin on viability and GSH levels in primary cortical neurons. Viability (A) and GSH levels (D) of 7 day rat primary cortical neurons treated with increasing doses of the peroxynitrite donor SIN-1 for 6 hr and/or the neuroprotective flavonoid fisetin (10 μM) and/or the exogenous GSH source glutathione monoethyl ester (GEE) in the absence or presence of the glutathione synthesis inhibitor buthionine sulfoximine (BSO) (1 mM). (B) Viability of 7 day rat primary cortical neurons treated with 1 mM SIN-1 for 6 hr alone or in the presence of 1 mM uric acid or 25 μM FeTPPS. (C) Viability of 7 day rat primary cortical neurons treated with 250 μM peroxynitrite for 24 hr alone or following pretreatment for 30 min or 24 hr with 10 μM fisetin. In the case of the 24 hr pretreatment, fisetin was removed from the cells before the addition of peroxynitrite. Cell viability was measured by the MTT assay. GSH levels were measured using monochlorobimane. Similar results were obtained in three independent experiments. * indicates significantly different (p < 0.05) from control; # indicates significantly different from SIN-1 or peroxynitrite alone; & indicates significantly different from BSO alone; % indicates significantly different from fisetin + SIN-1 treatment.

Figure 2

Analysis of ERK1/2 phosphorylation in primary cortical neurons treated with SIN-1, fisetin and PD98059. (A) 7 day primary cortical neurons in 35 mm dishes were left untreated (Ctl) or were treated for 6 hr with 1 mM SIN-1, 10 μM fisetin and/or 40 μM PD98059 as indicated in the figure. Cells were pre-treated with fisetin or PD98059 for 15 min before the addition of SIN-1. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho-ERK along with antibodies to total ERK2 demonstrating no changes in overall protein levels. Similar results were obtained in three independent experiments. The average phosphoprotein signal from the blots was quantified by densitometry, normalized to total protein and plotted ± S.D. * indicates significantly different (p < 0.05) from control; # indicates significantly different from SIN-1 alone. (B) 7 day primary cortical neurons in 35 mm dishes were treated for 15 min to 6 hr with 1 mM SIN-1. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho-ERK along with antibodies to total ERK2 demonstrating no changes in overall protein levels. Similar results were obtained in three independent experiments. Fis = Fisetin, PD = PD98059

Figure 3

The effects of SIN-1, fisetin and PD98059 on the transcription factors c-Myc and Nrf2. (A) Time course of the effects of SIN-1 on c-Myc phosphorylation. 7 day primary cortical neurons in 35 mm dishes were left untreated (Ctl) or were treated for 30 min to 6 hr with 1 mM SIN-1. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho-c-Myc along with antibodies to total c-Myc demonstrating no changes in overall protein levels. Similar results were obtained in three independent experiments. The average phosphoprotein signal from the blots was quantified by densitometry, normalized to total protein and plotted ± S.D. (B) Inhibition of c-Myc dephosphorylation by fisetin and PD98059. 7 day primary cortical neurons in 35 mm dishes were left untreated (Ctl) or were treated for 6 hr with 1 mM SIN-1, 10 μM fisetin and/or 40 μM PD98059 as indicated in the figure. Cells were pre-treated with fisetin or PD98059 for 15 min before the addition of SIN-1. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho-c-Myc along with antibodies to total c-Myc demonstrating no changes in overall protein levels. Similar results were obtained in three independent experiments. The average phosphoprotein signal from the blots was quantified by densitometry, normalized to total protein and plotted ± S.D. (C) The SIN-1-dependent decrease in Nrf2 is prevented by fisetin but not PD98059. 7 day primary cortical neurons in 35 mm dishes were left untreated (Ctl) or were treated for 6 hr with 1 mM SIN-1, 10 μM fisetin and/or 40 μM PD98059 as indicated in the figure. Cells were pre-treated with fisetin or PD98059 for 15 min before the addition of SIN-1. Nuclei were prepared and equal amounts of nuclear protein were analyzed by SDS-PAGE and immunoblotting with antibodies to Nrf2 along with antibodies to actin as a loading control. The average Nrf2 signal from the blots was quantified by densitometry, normalized to actin and plotted ± S.D. * indicates significantly different (p < 0.05) from control; # indicates significantly different from SIN-1 alone. Fis = Fisetin, PD = PD98059

Figure 4

The opposing effects of SIN-1 and fisetin on the levels of the two subunits of glutamate cysteine ligase (GCL), the rate limiting enzyme in GSH biosynthesis. 7 day primary cortical neurons in 35 mm dishes were left untreated (Ctl) or were treated for 6 hr with 1 mM SIN-1, 10 μM fisetin or 10 μM fisetin + 1 mM SIN-1. Cells were pre-treated with fisetin 15 min before the addition of SIN-1. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to GCLC or GCLM along with antibodies to actin as a loading control. The average GCLC or GCLM signal from the blots was quantified by densitometry, normalized to actin and plotted ± S.D. * indicates significantly different (p < 0.05) from control; # indicates significantly different from SIN-1 alone. Fis = Fisetin

Figure 5

Inhibitors of the Ras-ERK cascade increase GSH biosynthesis in unstressed primary cortical neurons. (A) 7 day primary cortical neurons in 35 mm dishes were left untreated (Ctl) or were treated for 6 hr with 100 μM of the Raf-1 kinase inhibitor RKI or 10 μM of the MEK inhibitor U0126. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho-ERK and ERK2, phospho-c-Myc and c-Myc, Nrf2, GCLC, GCLM and actin. Similar results were obtained in three independent experiments. The average phosphoprotein or Nrf2, GCLC or GCLM signal from the blots was quantified by densitometry, normalized to total protein or actin, as indicated in the figure, and plotted ± S.D. (B) 7 day primary cortical neurons were left untreated (Ctl) or were treated for 6 hr with 100 μM of the Raf-1 kinase inhibitor RKI or 10 μM of the MEK inhibitor U0126. GSH levels were measured using monochlorobimane and plotted relative to untreated, control cells. * indicates significantly different (p < 0.05) from control.

Figure 6

Model showing the opposing effects of SIN-1 and fisetin on primary cortical neurons. In stressed cells, treatment with the peroxynitrite donor, SIN-1, leads to the hyperphosphorylation of ERK1/2 by MEK, which results in a decrease in c-Myc phosphorylation, a decrease in GCLC/GCLM levels and a decrease in intracellular GSH. SIN-1 treatment also causes a decrease in Nrf2 levels which contributes to the decrease in GSH levels. Pretreatment of the cells with fisetin prevents these SIN-1-mediated alterations and results in the maintenance of cellular GSH. Large arrows indicate activation, “T” lines indicate inhibition, small arrows indicate direction of change of specific protein phosphorylation events (ERK and c-Myc) or levels of GCLC/GCLM and GSH.