Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells

Abstract

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.

In vivo demonstration that α-synuclein oligomers are toxic

“…LUHMES cells were used for tissue culture experiments. They are a conditionally immortalized human neuronal cell line and were a gift from M. Leist and D. Schols (28). The LUHMES cells were maintained as described previously under a proliferative state with FGF-2 or under neuronal differentiation conditions (28).…”

“…To test if the mutants that favor oligomer formation compared with the mutants that form fibrils are more quickly toxic in a cellbased system, we investigated cell death in a human mesencephalic immortalized neuronal cell line [Lund human mesencephalic (LUHMES) cells (28)] infected with the α-syn lentivirus. The percentage of activated caspase-3/DAPI-positive cells was analyzed.…”

Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes

“…For proliferation, cells were cultured in Advanced Dulbecco's modified Eagle's medium/F12 (Advanced DMEM/F12, Life Technologies GmbH, Darmstadt, Germany) supplemented with 1 × N2 supplement (Life Technologies), 2 mM l-glutamine (Biochrom, Berlin, Germany) and 40 ng/mL recombinant human basic fibroblast growth factor (FGF, R&D Systems, Wiesbaden-Nordenstadt, Germany) at 37 • C in a humidified 95% air, 5% CO 2 atmosphere. In accordance to the published protocol [25,27], cell differentiation was initiated 24 h after seeding the cells at a density of 45,000 cells per cm 2 by replacing the proliferation medium with differentiation medium consisting of Advanced DMEM/F12 containing 1 × N2 supplement, 2 mM l-glutamine, 1 g/mL tetracycline (Sigma-Aldrich), 1 mM dibutyryl cyclic adenosine monophosphate sodium salt (cAMP, Sigma-Aldrich) and 2 ng/mL recombinant human glial cell-derived neurotrophic factor (GDNF, R&D Systems). After 48 h of differentiation, cells were trypsinized and seeded on pre-coated dishes in a defined density (150,000 cells/cm 2 ) in differentiation medium.…”

Studying neurodegenerative diseases in culture models

“…27 Differentiated LUHMES cells showed a high degree of dopaminergic phenotype, including release of dopamine and neuronal electric properties. 28,29 The LUHMES cell line has been widely used to study dopamine-related cell death mechanisms. 27,29,30 A drawback of this cell line is that classical transfection methods showed very low transfection efficiency.…”

“…28,29 The LUHMES cell line has been widely used to study dopamine-related cell death mechanisms. 27,29,30 A drawback of this cell line is that classical transfection methods showed very low transfection efficiency. Thus, a lentiviral approach to efficiently transfect these cells is necessary.…”