Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway

Abstract

Models of Parkinson's disease (PD) based on selective neuronal death have been used to study pathogenic mechanisms underlying nigral cell death and in some instances to develop symptomatic therapies. For validation of putative neuroprotectants, a model is desirable in which the events leading to neurodegeneration replicate those occurring in the disease. We developed a human in vitro model of PD based on the assumption that dysregulated cytoplasmic dopamine levels trigger cell loss in this disorder. Differentiated human mesencephalic neuron-derived cells were exposed to methamphetamine (METH) to promote cytoplasmic dopamine accumulation. In the presence of elevated iron concentrations, as observed in PD, increased cytosolic dopamine led to oxidative stress, c-Jun N-terminal kinase (JNK) pathway activation, neurite degeneration, and eventually apoptosis. We examined the role of the mixed-lineage kinases (MLKs) in this complex degenerative cascade by using the potent inhibitor 3,9-bis[(ethylthio)methyl]-K-252a (CEP1347). Inhibition of MLKs not only prevented FeCl2+/METH-induced JNK activation and apoptosis but also early events such as neurite degeneration and oxidative stress. This broad neuroprotective action of CEP1347 was associated with increased expression of an oxidative stress-response modulator, activating transcription factor 4. As a functional consequence, transcription of the cystine/glutamate and glycine transporters, cellular cystine uptake and intracellular levels of the redox buffer glutathione were augmented. In conclusion, this new human model of parkinsonian neurodegeneration has the potential to yield new insights into neurorestorative therapeutics and suggests that enhancement of cytoprotective mechanisms, in addition to blockade of apoptosis, may be essential for disease modulation.

Figure 1.

Iron (FeCl₂) significantly potentiates METH toxicity in differentiated LUHMES cells. A, Differentiated LUHMES cells were treated with 1 mm METH or 75 μm FeCl₂ alone or FeCl₂ plus METH (Fe/METH) for 5 d, and neurite integrity was qualitatively assessed by both β-III-tubulin and MAP2 immunocytochemistry. B, Cell viability was measured in cultures exposed to 50-1000 μm METH for 5 d by a battery of cell death/viability assays. C, Potentiation of METH toxicity was examined in cultures treated with 75 μm FeCl₂, 1 mm METH, or both (Fe/METH). Cell survival was assessed after 72 h by visual inspection of calcein AM-positive cells. D, A set of Fe²⁺/METH-treated cells (F/M) was cotreated for 72 h with 250 μm DM or 100 μm α-MPT, and cell viability was quantified by counting the number of calcein AM-positive cells. All data are expressed as the mean ± SEM of quadruplicate determinations and are shown as a percentage of control cultures. Error bars represent SEM.

Figure 2.

Fe²⁺/METH leads to progressive neurite degeneration, which is significantly attenuated by CEP1347. A, LUHMES cells were treated with Fe²⁺/METH for 24-72 h, fixed, and immunoprocessed for β-III-tubulin. Cells were also cotreated with Fe²⁺/METH in the presence of 250 nm CEP1347 for 72 h. B, Neurite mass and cell viability were quantified in cultures immunostained for β-III-tubulin 0-72 h after exposure to Fe²⁺/METH, using a Cellomics ArrayScan instrument. Neuritic β-III-tubulin immunofluorescence was used as a measure of neurite mass. Number of cells, as assessed by neurons with normal nuclear morphology, was used as an indicator of cell survival. Data are expressed as mean ± SEM of quadruplicate determinations and are shown as a percentage of control. C, LUHMES cells were exposed to Fe²⁺/METH for 72 h in the presence or absence of 250 nm CEP1347, and neurite mass was quantified with the Cellomics ArrayScan. Values are expressed as mean ± SEM of quadruplicate determinations and are shown as a percentage of control. D, LUHMES cultures were treated with 250 nm CEP1347 (CEP) 6-48 h after Fe²⁺/METH (F/M) addition. All cultures were fixed and immunostained for β-III-tubulin 72 h after the initial insult. Error bars represent SEM.

Figure 3.

Fe²⁺/METH induces activation of the JNK signaling pathway, which is blocked by CEP1347. A, LUHMES cells were exposed to Fe²⁺/METH for 6-60 h, and the induction of JNK pathway intermediates was assessed by Western blotting using antibodies recognizing the phosphorylated forms of MKK4 (p-MKK4), JNK (p-JNK), and c-Jun (p-c-Jun). GAPDH was used as a loading control. B, LUHMES cells were cotreated with Fe²⁺/METH and 250 nm CEP1347 for 36 h, and the levels of p-MKK4, p-JNK, and p-c-Jun were measured by Western blotting. Blots shown represent three independent experiments.

Figure 4.

The MLK inhibitor CEP1347 rescues LUHMES cells and primary rat ventral mesencephalic dopaminergic neurons from Fe²⁺/METH-induced toxicity. A, LUHMES cells were treated with Fe²⁺/METH for 72 h in the presence of 50-1000 μm CEP1347, and cell viability was assessed by counting the number of calcein AM-positive cells and by measuring MTT reduction capacity. B, Micrographs showing cultures treated with Fe²⁺/METH in the presence or absence of 250 nm CEP1347 stained with calcein AM. C, Primary rat ventral mesencephalic cultures were treated with METH alone or in the presence of 100-1000 μm CEP1347, and dopaminergic cell survival was assessed by counting the number of TH-positive neurons. Values are expressed as mean ± SEM of quadruplicate determinations and are shown as a percentage of control cultures. C, CEP1347 was added to LUHMES cells either 1 h before or 2-72 h after Fe²⁺/METH addition. The toxicity of Fe²⁺/METH was measured after 72 h by quantification of LDH release and expressed as a percentage of neuroprotection compared with control cultures. All data are expressed as the mean ± SEM of quadruplicate determinations. D, Caspase-3-like enzyme activity in LUHMES cells treated with Fe²⁺/METH in the presence or absence of 250 nm CEP1347 was quantified for the ability to cleave a fluorogenic tetrapeptide, Ac-DEVD-AFC, which is a substrate for caspase-3, caspase-6, caspase-7, caspase-8, and caspase-10, after 6-60 h of drug treatment. The fluorescence emitted by cleavage of the substrate was quantified during a 20 min incubation period by kinetic fluorimetric measurements. Values were standardized to micrograms of protein, expressed as mean ± SEM of quadruplicate determinations, and shown as a percentage of control cultures. Error bars represent SEM.

Figure 5.

Activation of AKT and its role in the neuroprotective effects of CEP1347. A, Protein levels of phosphorylated AKT (Thr³⁰⁸) were measured by Western blotting in whole-cell lysates from LUHMES cultures treated with Fe²⁺/METH for 6-36 h in the presence or absence of 250 nm CEP1347. B, Levels of p-AKT were measured by Western blotting after 24 h of treatment with CEP1347, Fe²⁺/METH with CEP1347 (F/M + CEP), or Fe²⁺/METH with CEP1347 in the presence or absence of a 10 μm concentration of the PI3 kinase inhibitor LY294002. For A and B,β-actin was used as a loading control. Blots shown are representative of three independent experiments, and numbers under the blots correspond to densitometric quantification of band densities. C, LUHMES cells were treated with Fe²⁺/METH in the presence or absence of 250 nm CEP1347 and 10 μm LY294002. MTT reduction was used to assess cell survival after 72 h. Values are expressed as mean ± SEM of quadruplicate determinations and are shown as a percentage of control. D, To exclude the possibility that LY294002 may be inefficacious in blocking PI3K at later time points during >6 h of CEP1347 exposure, the inhibitor was added 6-60 h after the addition of Fe²⁺/METH and CEP1347. Viability was assayed after 72 h by measuring the release of LDH into the medium. Values are expressed as mean ± SEM of quadruplicate determinations and are shown as a percentage of control. Error bars represent SEM.

Figure 6.

Fe²⁺/METH triggers oxidative stress, which is blocked by CEP1347. A, LUHMES cells were exposed to Fe²⁺/METH (F/M) and varying concentrations of antioxidants for 72 h. Results for treatment with 250 nm CEP1347 (as a positive control), 200 U/ml SOD, 1 μm MnTBAP, 250 μm DM, 200 U/ml catalase, and 100 μm NAC are shown. Cell survival was assessed by counting calcein AM-positive cells. Data are expressed as mean ± SEM of quadruplicate determinations and are shown as a percentage of control. B, LUHMES cells were treated with Fe²⁺/METH with or without 250 nm CEP1347, and the levels of MDA, a lipid peroxidation product, were measured using a Bioxytech MDA-596 kit from OxisResearch. C, The effect of 1 mm METH on H₂O₂ production was examined over 24 h using the Bioxytech H₂O₂-560 kit from OxisResearch (see Materials and Methods). FeCl₂ was not added in the cultures for H₂O₂ measurements because of interference of Fe²⁺ with the assay reaction. D, Levels of H₂O₂ and superoxide (), the latter of which was assayed fluorimetrically by measuring the oxidation of dihydroethidium to fluorescent ethidium, were measured after 12 h (H₂O₂) or 36 h () of Fe²⁺/METH treatment in the presence or absence of 250 nm CEP1347. For B and C, values were standardized to protein concentration and expressed as mean ± SEM of quadruplicate determinations. Data are shown as a percentage of control. Error bars represent SEM.

Figure 7.

CEP1347 increases cellular antioxidant capacity. A, LUHMES cells were treated with Fe²⁺/METH in the presence or absence of 250 nm CEP1347 for 24 h, and total intracellular glutathione levels were measured by a photometric assay. Values were standardized to protein concentration, normalized to control cultures, and expressed as mean ± SEM of quadruplicate determinations. B, LUHMES cells were treated with 250 nm CEP1347 for 6-48 h, and GSH levels were measured. C, Using qPCR, mRNA levels of the transcription factor ATF4 were assessed after 6 or 12 h of Fe²⁺/METH exposure in the presence or absence of 250 nm CEP1347. Cells exposed to 250 nm CEP1347 were also tested. Values were standardized to GAPDH mRNA and are expressed as a ratio of the gene of interest to GAPDH mRNA ± SEM of quadruplicate determinations. D, mRNA levels of the Cys-Glu transporter were also measured by qPCR after 6 or 12 h of drug exposure. E, mRNA levels of the Gly transporter were assessed as in D. F, Protein levels of ATF4 were examined by Western blotting 12 h after exposure to 250 nm CEP1347, Fe²⁺/METH, Fe²⁺/METH in the presence of CEP1347, or Fe²⁺/METH in the presence of CEP1347 and a 10 μm concentration of the PI3 kinase inhibitor LY294002. Blots shown are a representation of three independent experiments. G, [¹⁴C] cystine uptake was measured in LUHMES cells treated for 16 h with Fe²⁺/METH in the presence or absence of 250 nm CEP1347. Values were standardized to protein concentration, normalized to control, and expressed as mean ± SEM of quadruplicate determinations. Error bars represent SEM.