Toxicity, recovery, and resilience in a 3D dopaminergic neuronal in vitro model exposed to rotenone

Abstract

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. In addition, to study cellular resilience, cells were re-exposed to rotenone after wash-out and recovery period. 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 14, 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.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2250-8) contains supplementary material, which is available to authorized users.

Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity

“…Here, we studied the metabolic incorporation of an azidetagged sugar precursor (Ac 4 ManNAz) as Az-Sia into the glycocalyx of mature human neurons (LUHMES). These cells (Scholz et al 2011) are a frequently used model for human neurotoxicity (Delp et al 2018b(Delp et al , 2019Efremova et al 2017;Ganjam et al 2019;Harris et al 2018;Krug et al 2013;Singh et al 2018;Skirzewski et al 2018;Stiegler et al 2011;Tong et al 2018). We asked the question on whether the sialylation capacity can be used as an early functional readout of neurotoxicity.…”

The conserved DNMT1 dependent methylation regions in human cells are vulnerable to environmental rotenone

“…We treated human cell line HEK293 with rotenone (200nm) for 24h. This dose was chosen based on previous reports in HEK293 and other neuronal cell models (Harris et al 2018, Orth et al 2003, Teixeria et al 2018). Rotenone treatment had a substantial effect on the expression levels of over 2,000 genes (≥1.5 fold, FDR≤0.05).…”

The conserved DNMT1-dependent methylation regions in human cells are vulnerable to neurotoxicant rotenone exposure

“…These brain regions include the cerebellum, spinal cord, striatum, and basal ganglia. We treated human cell line HEK293 with rotenone (200 nm) for 24 h. This dose was chosen based on previous reports in HEK293 and other neuronal cell models [28,56,73]. Rotenone treatment had a substantial effect on the expression levels of over 2000 genes (≥ 1.5-fold, FDR ≤ 0.05).…”