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3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects.

ABSTRACT: Current neurotoxicity testing and the study of molecular mechanisms in neurodegeneration in vitro usually focuses on acute exposures to compounds. 3D Lund human mesencephalic (LUHMES) cells allow long-term treatment or pulse exposure in combination with compound washout to study delayed neurotoxic effects as well as recovery and neurodegeneration pathways. In this unit we describe 3D LUHMES culture and characterization. Characterization of the model involves immunocytochemistry, flow cytometry, and qPCR measurements. Studying the delayed effects of compounds is more relevant to human exposures and neurodegenerative diseases with a strong genetic or environmental component. Most assays for molecular endpoints have been developed for monolayer cell culture and therefore need to be adapted for 3D models. In this unit, we further describe toxicological assays for molecular endpoints such as ATP levels, mitochondrial viability, and neurite outgrowth, which have been adapted for use in 3D LUHMES cultures. © 2017 by John Wiley & Sons, Inc.

SUBMITTER: Harris G

PROVIDER: S-EPMC5674809 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects.

Harris Georgina G Hogberg Helena H Hartung Thomas T Smirnova Lena L

Current protocols in toxicology 20170804

Current neurotoxicity testing and the study of molecular mechanisms in neurodegeneration in vitro usually focuses on acute exposures to compounds. 3D Lund human mesencephalic (LUHMES) cells allow long-term treatment or pulse exposure in combination with compound washout to study delayed neurotoxic effects as well as recovery and neurodegeneration pathways. In this unit we describe 3D LUHMES culture and characterization. Characterization of the model involves immunocytochemistry, flow cytometry, ...[more]

PMID: 28777440

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Project description: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

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3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects.

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3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects.

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