Article Figures & Data
Figures
- Fig. 1.
MIT is neurotoxic in vitro.A–D, Phase–contrast micrographs of rat cortical cultures 24 hr after being treated for 10 min with either vehicle (A), 100 μm MIT (B), 10 μm TPEN (C), or 100 μm MIT plus 10 μm TPEN (D). Note the relative absence of phase–bright (live) neurons in B and the neuroprotective actions of TPEN in D. E, Concentration–toxicity relationship for MIT in control mixed cultures (Neurons/Glia) and in sister cultures that had been treated 72 hr earlier with kainic acid (1 mm, 24 hr) to remove the neuronal component (Glia). MIT results in a large increase in LDH release (an index of cell death) in the mixed but not in the glial cultures. LDH release induced by a 1 hr exposure to 200 μm NMDA (a selective neuronal toxin) is included for comparison. Values represent the mean ± SD for a total of six experiments in the mixed cultures and three experiments in the kainate-treated cells; ∗∗p < 0.01; ∗∗∗p < 0.001. The inset shows a representative experiment, in triplicate (mean ± SD), performed on primary mouse astrocytes. A 10 min exposure to 100 μmMIT was not toxic to the cells. Total LDH in the culture was measured after cell lysis; ∗∗∗p < 0.001.F, LDH activity measured with known concentrations of the enzyme alone or in the continuous presence of 100 μmMIT. No differences were observed between the two standard curves. Values represent the mean ± SD of three independent measurements.
- Fig. 2.
Role of Zn2+ in MIT toxicity.A, Cortical cultures were exposed to 100 μm MIT (10 min) in the absence or presence of various concentrations of TPEN. LDH release for each experimental group was normalized to that generated by MIT alone (100% relative toxicity) in this and subsequent figures. Note that the toxicity after MIT exposure decreases as a function of TPEN concentration. Values represent the mean ± SEM (n = 4); ∗∗p < 0.01; ∗∗∗p < 0.001; significantly different from MIT alone. The insetconfirms the neuroprotective actions of 1 μm TPEN, as determined by cell counting; ∗∗∗p < 0.001.B, The neuroprotective action of 1 μm TPEN was eliminated by preincubating the chelating agent with equimolar zinc (ZnCl2) but not iron (FeSO4); ∗p < 0.05; ∗∗p < 0.01 (n = 4). C, Similar toB above, except that neurons were killed by 100 μm DTDP instead of MIT (Aizenman et al., 2000; McLaughlin et al., 2001) (n = 3).
- Fig. 3.
MIT toxicity involves ERK activation.A, Immunoblots of cell extracts from cortical cultures harvested at various time points after a 10 min exposure to 100 μm MIT. Proteins were separated by SDS-PAGE and probed with antibodies specific to the phosphorylated and nonphosphorylated forms of both p38 and p44/42 ERK. Note the absence of phosphorylated p38 (Ph-p38) at all time points and the early increase in phosphorylated ERK (Ph-ERK) after MIT exposure. The MEK inhibitor U0126 (10 μm) and the zinc chelator TPEN (1 μm) completely blocked ERK phosphorylation (30 min). Similar results were obtained in three additional experiments. Aniso, Anisomycin;Con, control. B, MIT toxicity was not blocked by the p38 inhibitor SB239063 (20 μm) but was significantly inhibited by the MEK inhibitors U0126 (10 μm) and PD98059 (40 μm). Results represent the mean ± SEM of three to four independent experiments; ∗p < 0.05. C, A lack of p38 involvement in MIT toxicity was confirmed by a lack of enhancement in potassium channel currents 3–4 hr after a 10 min MIT (100 μm) exposure (compare with McLaughlin et al., 2001). Results represent the mean ± SD current density (n = 6, 11) for potassium currents evoked in voltage-clamped cortical neurons by stepping the voltage to −10 mV from a holding voltage of −70 mV. Con, Control.Insets, Examples of whole-cell potassium currents obtained in two separate cortical neurons ∼3 hr after a 10 min exposure to vehicle or MIT. Currents were evoked by a series of steps to 35 mV from a holding voltage of −70 mV. D, Lack of neuroprotection against MIT by the following agents: TEA (10 mm), high extracellular potassium (25 mm), cyclohexamide (CHX, 3.5 μm), and BAF (20 μm); data represent the mean ± SEM (n = 4–9).
- Fig. 4.
MIT toxicity and MIT-induced ERK activation requires 12-LOX activity. A, MIT (100 μm) toxicity was significantly inhibited by the 12-LOX inhibitor baicalein (20 μm) and by the less-specific LOX inhibitor AA861 (1 μm). Results represent the mean ± SEM (n = 3); ∗∗∗p < 0.001.B, Immunoblots demonstrate that MIT-induced ERK activation is blocked by baicalein. Baicalein had no effect on ERK activation when added alone (data not shown). Similar results were observed in a total of three independent experiments.Phospho-ERK, Phosphorylated ERK.C–F, 12-LOX immunostaining in control cultures (C) and 5 min after a 10 min exposure to 100 μm MIT alone (D) or in the presence of either 20 μm baicalein (E) or 1 μm TPEN (F). LOX activation is usually accompanied by its translocation to the cell membrane.
- Fig. 5.
MIT induces a decrease in GSH activity. Cultures were exposed to 100 μm MIT for 10 min and assayed for cellular GSH activity 5 min later. MIT induces a decrease in GSH levels, which can be prevented by coexposure to the cysteine reagent NAC (1 mm) but not by the antioxidant trolox (100 μm) or by TPEN (1 μm) (mean ± SEM;n = 3–4); ∗∗p < 0.01.
- Fig. 6.
Effect of antioxidants and PARP inhibitors on MIT-induced toxicity. A, MIT toxicity was abrogated by including NAC (1 mm), trolox (100 μm), and GSH (1 mm) before, in conjunction with, and after a 10 min MIT (100 μm) treatment. NAC, but not trolox, lost its neuroprotective properties when included in the postexposure period only (post). The PARP inhibitors NU1025 (5 μm) and DPQ (1 μm) were also protective. Results represent the mean ± SEM for three to four experiments; ∗∗p < 0.01; ∗∗∗p < 0.001. B, Immunoblots demonstrating that ERK phosphorylation after MIT exposure could be abolished by the NAC treatment but not by trolox (before exposure, during exposure, and after exposure). The effect of NAC was absent when the antioxidant was included in the postexposure period only. Trolox, NAC, and NU1025 had no effect on ERK activation when added alone (data not shown). Similar results were obtained in a total of three independent experiments. These results suggest that ROS production and PARP activation occur downstream from ERK phosphorylation. Phospho-ERK, Phosphorylated ERK.
- Fig. 7.
NADPH oxidase inhibitors are protective against MIT toxicity. A, MIT (100 μm) toxicity was significantly inhibited by the NADPH inhibitors AEBSF (100 μm) and DPI (100 nm). Results represent the mean ± SEM (n = 3); ∗p< 0.05. B, Immunoblots demonstrating that MIT-induced ERK activation was not blocked by AEBSF or DPI. Neither of these compounds had an effect on ERK phosphorylation when added alone (data not shown). Similar results were observed in a total of three independent experiments. Phospho-ERK, Phosphorylated ERK.
- Fig. 8.
MIT induces ERK-dependent DNA damage. Four hours after a 10 min exposure to 100 μm MIT, cultures were stained for TUNEL and counterstained with thionin. Using this procedure, nuclei with damaged DNA are stained darkbrown (arrowheads in B), and cells are stained purple. A, Control.B, MIT-treated cultures. C, MIT-treated cultures in the presence of the MEK inhibitor U0126 (10 μm). D, MIT-treated cultures in the presence of the antioxidant trolox (100 μm). Note the absence of TUNEL staining in A, C, andD.
