Sustained extracellular signal-regulated kinase activation by 6-hydroxydopamine: implications for Parkinson's disease

Abstract

Although the toxin 6-hydroxydopamine (6-OHDA) is utilized extensively in animal models of Parkinson's disease, the underlying mechanism of its toxic effects on dopaminergic neurons is not completely understood. We examined the effects of 6-OHDA on the CNS-derived tyrosine hydroxylase expressing B65 cell line, with particular attention to the regulation of the extracellular signal-regulated protein kinases (ERK). 6-OHDA elicited a dose-dependent cytotoxicity in B65 cells. Toxic doses of 6-OHDA also elicited a biphasic pattern of ERK phosphorylation with a prominent sustained phase, a pattern that differed from that observed with hydrogen peroxide (H(2)O(2)) treatment. 6-OHDA-elicited ERK phosphorylation was blocked by PD98059, an inhibitor of the upstream mitogen activated protein kinase kinase (MEK) that phosphorylates and activates ERK. PD98059 also conferred protection against 6-OHDA cytotoxicity, but did not affect H(2)O(2) toxicity in B65 cells. These results suggest that ERK activation plays a direct mechanistic role in 6-OHDA toxicity, rather than representing a protective compensatory response, and raise the possibility that abnormal patterns of ERK activation may contribute to dopaminergic neuronal cell death.

Fig. 1

Cytotoxicity of 6-OHDA and H₂O₂ in B65 cells. B65 cells were exposed to (a) 6-OHDA or (b) H₂O₂ for 18–20 h. Cell injury was determined by measuring LDH activity released into the media (○) or by altered MTS metabolism (●). Values were normalized to total LDH activity or the A₄₉₀ of vehicle treated control cells, respectively, as described in the Materials and methods section. Each value represents the mean of triplicate wells ± SE. Each experiment was performed independently at least three times. Representative plots are illustrated.

Fig. 2

Dose–response study of ERK activation in B65 cells. Cells were treated with the indicated μM concentrations of 6-hydroxydopamine (6-OHDA), H₂O₂ (H₂O₂) or vehicle (0.05% ascorbate for 6-OHDA, water for H₂O₂) for 18 h. The cells were then lysed and equal amounts of protein (20 μg) were subjected to immunoblot analysis using an antibody specific for activated dually phosphorylated ERK (top, P-ERK). The blots were then stripped and reprobed with antibody against total ERK (bottom, T-ERK). Blots depicted are representative of eight independent experiments.

Fig. 3

Kinetics of ERK activation in B65 cells in response to 6-OHDA and H₂O₂. (a) Cells treated with 500 μM 6-hydroxydopamine (6-OHDA) or 1 mM H₂O₂ (H₂O₂) for the indicated times (minutes) were lysed and equal amounts of protein (50 μg) were subjected to immunoblot analysis using antibody against the activated form of ERK (top, P-ERK). The blots were then stripped and reprobed with antibody against total ERK (bottom, T-ERK). Lanes designated with * were harvested following an overnight (16 h) incubation. Lanes designated with – were treated with the appropriate vehicle (0.05% ascorbate for 6-OHDA, water for H₂O₂). (b) Cells were treated with either 1000 or 500 μM 6-OHDA or 1 mM H₂O₂ for the indicated time (hours) then subjected to immunoblot analysis as in (a). (c) Immunoblots from cells treated with 1000 mM 6-OHDA were subjected to densitometric analysis, and the data for the ERK1 (top panel) and ERK2 (bottom panel) isoforms from a representative time-course is shown. Closed circles (●) represent the relative band intensity of phosphorylated ERK/total ERK elicited by 6-OHDA; open circles (○) represent the phosphorylated ERK/total ERK ratio observed in vehicle-treated cells. Cells treated with only fresh DH10 exhibited a pattern of ERK phosphorylation similar to that seen with the vehicle controls. (d) Immunoblots from cells treated with 500 μM 6-OHDA and 1 mM H₂O₂ were subjected to densitometric analysis, and the data for the ERK1 (top panel) and ERK2 (bottom panel) isoforms from a representative time-course is shown. Closed circles represent the relative band intensity elicited by 6-OHDA while open circles represent the relative band intensity elicited by H₂O₂. Data are expressed as the ratio of phosphorylated ERK to total ERK elicited by the oxidizing agents (pERK/tERK-OX) normalized to the ratio of phosphorylated ERK to total ERK elicited in vehicle treated cells (pERK/tERK-V). Data shown in panels A–D are representative compilations of 10 independent time-course experiments.

Fig. 4

Pulse-chase studies comparing ERK phosphorylation and cytotoxicity. (a) Cells were pulsed with 1 mM 6-hydroxydopamine (6-OHDA) for the indicated times (hours) at which time 6-OHDA was removed. The cells were washed with DH10 and then incubated in fresh DH10 for a total incubation time of 24 h. Immunoblot analysis of cell lysates was performed as described above. Cells treated with vehicle did not exhibit increased phosphorylation at any time point (24 h time point shown as an example). Data shown are representative of four independent experiments. (b) Cells were pulsed with various concentrations of 6-OHDA for the indicated times (2 h: ●, 4 h: ○, 6 h: ■, 22 h: □), washed and placed in fresh DH10 for a total incubation time of 22 h. Cell injury was determined using the MTS viability assay. Each value represents the mean of triplicate wells ± SEM. The experiment was performed independently three times, and a representative plot is illustrated.

Fig. 5

Effect of the MEK inhibitor PD98059 upon 6-OHDA mediated ERK phosphorylation. B65 cells treated for 20 h with 500 mM 6-hydroxydopamine (6-OHDA) in the presence (+) or absence (−) of 50 μM PD98059 were lysed and equal amounts of protein (50 μg) were subjected to immunoblot analysis using antibody against the activated form of ERK (top, P-ERK). The blots were then stripped and reprobed with antibody against total ERK (bottom, T-ERK).

Fig. 6

Effect of PD98059 on 6-OHDA-mediated cytotoxicity. Following overnight exposure of B65 cells to 655 mM 6-OHDA in the absence (white bar) or presence (hatched bar) of 50 mM PD98059, cytotoxicity was determined utilizing MTS (a) and LDH release (b) assays. Cells treated with 6-OHDA in the presence of 0.1% DMSO, the vehicle in which the inhibitor was dissolved (black bar), showed values similar to those lacking DMSO. PD98059 treatment had no effect upon the A₄₉₀ or total LDH values of ascorbate-treated control wells against which the other treatment conditions were normalized (A₄₉₀ values: 2.26 ± 0.06, untreated; 2.23 ± 0.08 PD98059 treated; total LDH values: 129 ± 27 IU/mL, untreated; 132 ± 23 IU/mL, PD 98059 treated). MTS data is a compilation of five independent experiments and LDH release data is a compilation of three independent experiments. Data are expressed as the mean ± SEM. Analysis by two-tailed Student's t-tests yielded p-values of <0.05 (*). (c) Following overnight exposure of B65 cells to 1 mM H₂O₂ in the absence (white bar) or presence (hatched bar) of 50 mM PD98059, cytotoxicity was determined utilizing a MTS assay. Cells treated with H₂O₂ in the presence of 0.1% DMSO (black bar), showed values similar to those lacking DMSO. MTS data is a compilation of five independent experiments, and is expressed as the mean ± SEM (p = 0.75, two-tailed Student's t-test).