Metabolic flux analysis in human dopaminergic neurons under toxicant stress

Abstract

Background: LUHMES cells are non-transformed conditionally immortalized human neuronal precursor cells which can be differentiated to dopaminergic neurons within six days.Methods: Proliferating or differentiated cells were used for experiments. Metabolomics analysis was performed using an LC-MS approach. For isotope labeling metabolic flux analysis cells were fed with 13C-glucose or 13C-glutamine. Subsequent GC-MS/MS analysis was used to quantify metabolite pools and fluxes. Flux maps resulted from modeling based on absolute concentrations and label incorporations into the central carbon metabolism (CCM)-metabolites.Results: Upon differentiation, LUHMES cells change their phenotype from precursor to fully differentiated dopaminergic neurons, e.g. shown by cell cycle arrest & expression of tyrosine hydroxylase. This change in phenotype is accompanied by a change in concentrations and fluxes of intermediates of the CCM. Using stable isotope labelled metabolite precursors, a flux map of the CCM of LUHMES was established for undifferentiated and differentiated cells. The metabolic flux analysis indicated that precursor cells have a stem cell-like metabolism, whereas differentiated cells acquire a neuronal-like metabolism. E.g. cells consumed drastically less glutamine when differentiated and began to secrete glutamate. Based on this set of background data, the metabolic impact of toxic chemicals can be described in high detail for various neuronal differentiation stages. E.g., substances that specifically inhibited neurite outgrowth, affected central carbon metabolism in a characteristic way. For the neurotoxicant MPP+ metabolic disturbances occurred long prior to other signs of damage. Compensatory flux regulations like increased utilization of glutamine were observed.