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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.

SUBMITTER: Harris G

PROVIDER: S-EPMC6063347 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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

Harris Georgina G Eschment Melanie M Orozco Sebastian Perez SP McCaffery J Michael JM Maclennan Richard R Severin Daniel D Leist Marcel M Kleensang Andre A Pamies David D Maertens Alexandra A Hogberg Helena T HT Freeman Dana D Kirkwood Alfredo A Hartung Thomas T Smirnova Lena L

Archives of toxicology 20180628 8

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. Afte ...[more]

PMID: 29955902

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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

Project description:BACKGROUND:Parkinson's disease (PD) is one of the neurodegeneration diseases characterized by the gradual loss of dopaminergic (DA) neurons in the substantia nigra region of the brain. Substantial evidence indicates that at the cellular level mitochondrial dysfunction is a key factor leading to pathological features such as neuronal death and accumulation of misfolded ?-synuclein aggregations. Autologous transplantation of healthy purified mitochondria has shown to attenuate phenotypes in vitro and in vivo models of PD. However, there are significant technical difficulties in obtaining large amounts of purified mitochondria with normal function. In addition, the half-life of mitochondria varies between days to a few weeks. Thus, identifying a continuous source of healthy mitochondria via intercellular mitochondrial transfer is an attractive option for therapeutic purposes. In this study, we asked whether iPSCs derived astrocytes can serve as a donor to provide functional mitochondria and rescue injured DA neurons after rotenone exposure in an in vitro model of PD. METHODS:We generated DA neurons and astrocytes from human iPSCs and hESCs. We established an astroglial-neuronal co-culture system to investigate the intercellular mitochondrial transfer, as well as the neuroprotective effect of mitochondrial transfer. We employed immunocytochemistry and FACS analysis to track mitochondria. RESULTS:We showed evidence that iPSCs-derived astrocytes or astrocytic conditioned media (ACM) can rescue DA neurons degeneration via intercellular mitochondrial transfer in a rotenone induced in vitro PD model. Specifically, we showed that iPSCs-derived astrocytes from health spontaneously release functional mitochondria into the media. Mito-Tracker Green tagged astrocytic mitochondria were detected in the ACM and were shown to be internalized by the injured neurons via a phospho-p38 depended pathway. Transferred mitochondria were able to significantly reverse DA neurodegeneration and axonal pruning following exposure to rotenone. When rotenone injured neurons were cultured in presence of ACM depleted of mitochondria (by ultrafiltration), the neuroprotective effects were abolished. CONCLUSIONS:Our studies provide the proof of principle that iPSCs-derived astrocytes can act as mitochondria donor to the injured DA neurons and attenuate pathology. Using iPSCs derived astrocytes as a donor can provide a novel strategy that can be further developed for cellular therapy for PD.

Project description:BackgroundThe rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing.MethodBASF Levasil® silica nanoparticles (16 and 85 nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells.ResultsFor all 2D exposures, significant (p < 0.002) increases in genotoxicity were observed (≥100 μg/mL) alongside cell viability decreases (p < 0.015) at doses ≥200 μg/mL (16 nm-SiO2) and ≥100 μg/mL (85 nm-SiO2). In contrast, 2D-equivalent exposures to the 3D models (≤300 μg/mL) caused no significant DNA damage or impact on cell viability. Further increasing dose to the 3D models led to probable air-liquid interface suffocation. Nanoparticle penetration / cell uptake analysis revealed no exposure to the live cells of the 3D model occurred due to the protective nature of the skin model's 3D cellular microarchitecture (topical exposures) and confounding barrier effects of the collagen cell attachment layer (in-medium exposures). 2D monocultured cells meanwhile showed extensive internalisation of both silica particles causing (geno)toxicity.ConclusionsThe results establish the importance of tissue microarchitecture in defining nanomaterial exposure, and suggest 3D in vitro models could play a role in bridging the gap between in vitro and in vivo outcomes in nanotoxicology. Robust exposure characterisation and uptake assessment methods (as demonstrated) are essential to interpret nano(geno)toxicity studies successfully.

Dataset Information

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

Publications

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

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