Factors affecting toxicant sensitivity of LUHMES-derived human neurons

The use of cell-based models in neurotoxicology and for neurodegenerative diseases is driven by limitations of animal data predictivity, legislative pressure and the in-creased availability of relevant cells. As all model systems cell-based in vitro models have their restrictions and limitations. For instance, the toxicological sensitivity of neuronal cells might be affected by genetic variability, differences in the metabolic state and the presence or absence of secondary cell types (e.g. astrocytes). The aim of this thesis was to characterize different factors affecting toxicity outcomes in neurons derived from a dopaminergic neuronal cell line (LUHMES: Lund human mesencephalic cells). In a first project, the conditionally immortalized cell line LUHMES was characterized with respect to changes in cellular metabolism occurring while differentiating from neuronal precursor cells to mature dopaminergic neurons. It was observed that changes in metabolic utilization and demands, i.e. the developmental state of the cells, correlate with an increase or decrease in toxicant sensitivity. The direction of the sensitivity change was defined for toxicants of different mode of action. In the second project, genomic stability and reproducibility within different subpopula-tions of this cell line were investigated. Notably, even though LUHMES have an intact set of chromosomes, a few passages in different environments (i.e. transfer to a dif-ferent lab and deposition at a cell bank) were sufficient to introduce differences be-tween subpopulations, as revealed by whole genome sequencing. These differences did not manifest as obvious genetic alterations, (i.e. larger chromosomal aberrations or mutations directly changing amino acids), but were rather founded in SNPs in non-coding regions or pleiotropic effects, which strongly influenced the toxicant sensitivity of the subpopulations. In the third project, the LUHMES cell line was used to discover stress pathways which increase cellular resilience against disturbance of the ubiquitin-proteasome system, one major hallmark of Parkinson’s disease (PD). NRF-1 was identified as a major transcription factor coordinating proteasomal recovery. Increased external glutathione (GSH) supply was found to increase NRF-1 expression and to allow cellular survival. By co-culturing LUHMES cells with astrocytes, it was observed that astro- 3 cytes are of great importance for neuronal thiol-supply and increase neuronal GSH, thereby reducing neuronal sensitivity for proteasomal inhibitors. In summary, these findings suggest that most model systems, used as basis for animal free methods, require more scientific characterization of robustness and resilience factors. By the characterization of these factors further insight into underlying mechanisms of physiological cell-cell-interactions, cellular resilience pathways as well as modes of toxicity can be discovered, and interpretation of toxicological data can be facilitated.publishe