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

ABSTRACT: Rotenone is a known neurotoxicant with both human and experimental data linking it to sporadic Parkinson's disease. We used RNAseq to identify global changes in gene expression and to identify vulnerable regions responsible to the rotenone treatment. Our goal was to determine if rotenone exposure alters chromatin mediated gene regulation mechanisms. We first explore their conservancy in the human genome. Our data show that our putative non-germline ASMs were in conserved regions of the human genome and located adjacent to genes vital for neuronal development and maturation. We next tested the hypothesized vulnerability of these regions by exposing human embryonic kidney cell HEK293 with the neurotoxicant rotenone for 24 h. Indeed,14 genes adjacent to our identified regions were differentially expressed from RNA-sequencing. We analyzed the base-resolution methylation patterns of the predicted non-germline ASMs at two neurological genes, HCN2 and NEFM, with potential to increase the risk of neurodegeneration. Both regions were significantly hypomethylated in response to rotenone.

ORGANISM(S): Homo sapiens

PROVIDER: GSE147617 | GEO | 2021/03/26

REPOSITORIES: GEO

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

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