Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells.
LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP(+)) toxicity at < or =5 microM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP(+) toxicity. Inhibition of de novo dopamine synthesis by alpha-methyl-l-tyrosine or 3-iodo-l-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP(+)-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP(+)-treated LUHMES.
Schildknecht
Stefan
S
University of Konstanz, Konstanz, Germany.
Pöltl
Dominik
D
Nagel
Daniel M
DM
Matt
Florian
F
Scholz
Diana
D
Lotharius
Julie
J
Schmieg
Nathalie
N
Salvo-Vargas
Alberto
A
Leist
Marcel
M
D015655:1-Methyl-4-phenylpyridinium / Q000008:administration & dosage / Q000633:toxicity*; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D050483:Dopamine Plasma Membrane Transport Proteins / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015227:Lipid Peroxidation; D020267:MPTP Poisoning; D008636:Mesencephalon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008781:Methyltyrosines / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism; D007470:Monoiodotyrosine / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors
C523964:3-iodotyrosine; D050483:Dopamine Plasma Membrane Transport Proteins; D008781:Methyltyrosines; D000255:Adenosine Triphosphate; C030855:3-iodo-alpha-methyltyrosine; D007470:Monoiodotyrosine; D005978:Glutathione; D015655:1-Methyl-4-phenylpyridinium