Acute and delayed transcriptomics changes in 3D LUHMES exposed to rotenone

To date, most in vitro toxicity testing has focused on acute effects of compounds at high concentrations. This testing strategy does not reflect real-life exposures, which might contribute to long-term disease outcome. We used a 3D-human dopaminergic in vitro LUHMES cell line model to determine whether effects of short-term rotenone exposure (100 nM, 24 h), are permanent or reversible. A decrease in complex I activity, ATP, mitochondrial diameter and neurite outgrowth were observed acutely. After compound removal, complex I activity was still inhibited, however, ATP levels were increased, cells were electrically active and aggregates restored neurite outgrowth integrity and mitochondrial morphology. We identified significant transcriptomic changes after 24 h which were not present 7 days after wash-out. Our results suggest that testing short-term exposures in vitro may capture many acute effects which cells can overcome, missing adaptive processes and long-term mechanisms. Additionally, to study cellular resilience, cells were re-exposed to rotenone after wash-out. Pre-exposed cells maintained higher metabolic activity than controls and presented a different expression pattern in genes previously shown to be altered by rotenone. NEF2L2, ATF4 and EAAC1 were downregulated upon single hit on day 15, but unchanged in pre-exposed aggregates. DAT and CASP3 were only altered after re-exposure to rotenone while TYMS and MLF1IP were downregulated in both single-exposed and pre-exposed aggregates. In summary, our study shows that a human cell-based 3D model can be used to assess cellular adaptation, resilience and long-term mechanisms relevant to neurodegenerative research. In this study acute and delayed effects of rotenone on transcriptome in 3D LUHMES were assessed by microarray. The goal was to characterize perturbations in gene expression immediately after exposure to rotenone and identify whether these perturbations persist after compound wash-out and recovery period LUHMES were differentiated in 3D and exposed to 50 or 100 nM rotenone on day 7 of differentiation for 12 or 24 hours. After the exposure half of the samples were collected for RNA isolation and in half of the samples rotenone was washed-out and cells were incubated for another 7 days until day 15. On day 15 the rest of the samples were collected for RNA isolation. Microarray gene expression analysis was performed on both groups of samples and normalized to the corresponding DMSO control in the group. Three replicates (wells) per sample were generated.