Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases

Schildknecht, Stefan; Karreman, Christiaan; Pöltl, Dominik; Efremova, Liudmila V.; Kullmann, Cornelius; Gutbier, Simon; Krug, Anne-Kathrin; Scholz, Diana; Gerding, Hanne R.; Leist, Marcel

doi:10.14573/altex.2013.4.427

Cited by 66 publications

(71 citation statements)

References 58 publications

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“…Schildknecht et al have recently reported a stable α-synuclein-overexpressing LUHMES subclone [32] . In their characterization, the increased amount of α-synuclein in LUHMES cells was well tolerated and no cytotoxicity was observed after overexpression of α-synuclein.…”

Section: Wwwnaturecom/apsmentioning

confidence: 99%

Cell-based assays for Parkinson's disease using differentiated human LUHMES cells

2014

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Aim: Lund human mesencephalic (LUHMES) cells can be differentiated to post-mitotic cells with biochemical, morphological and functional features of dopaminergic (DAergic) neurons. Given the limited scale of primary DAergic neuron culture, we developed differentiated LUHMES cell-based cytotoxicity assays for identifying neuroprotective agents for Parkinson's disease (PD). Methods: LUHMES cells were incubated in a differentiation medium containing cAMP and GDNF for 6 d, and then differentiated cells were treated with MPP + or infected with baculovirus containing α-synuclein. Cytotoxicity was determined by measuring intracellular ATP levels and caspase 3/7 activity in the cells. DAergic neuron-specific marker protein and mRNA levels in the cells were analyzed using Western blotting and RT-PCR, respectively. Results: LUHMES cells grew extensive neurites and became post-mitotic neuron-like cells during differentiation period, and three DAergic neuron markers TH, DAT and Nurr1 exhibited different expression profiles. MPP + dose-dependently reduced ATP levels in the cells with an IC 50 value of 65 μmol/L. MPP + (80 μmol/L) significantly increased caspase 3/7 activity in the cells. Both the CDK inhibitor GW8510 and the GSK3β inhibitor SB216763 effectively rescued MPP + -induced reduction of ATP levels with EC 50 values of 12 and 205 nmol/L, respectively. Overexpression of α-synuclein also significantly decreased intracellular ATP levels and increased caspase 3/7 activity in the cells. GW8510 and SB216763 effectively rescued α-synuclein overexpression-induced reduction of ATP levels, whereas GW8510, but not SB216763, ameliorated α-synuclein overexpression-induced increase of caspase 3/7 activity. Conclusion: MPP + -and α-synuclein overexpression-induced cytotoxicity of differentiated LUHMES cells may serve as good alternative systems for identifying neuroprotective compounds for PD.

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Section: Wwwnaturecom/apsmentioning

confidence: 99%

Cell-based assays for Parkinson's disease using differentiated human LUHMES cells

2014

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“…This human dopaminergic precursor cell line allows the generation of homogeneous, fully postmitotic dopaminergic neuron cultures. 28, 29 …”

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confidence: 99%

Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP+

et al. 2014

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Assessment of the network of toxicity pathways by Omics technologies and bioinformatic data processing paves the road toward a new toxicology for the twenty-first century. Especially, the upstream network of responses, taking place in toxicant-treated cells before a point of no return is reached, is still little explored. We studied the effects of the model neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) by a combined metabolomics (mass spectrometry) and transcriptomics (microarrays and deep sequencing) approach to provide unbiased data on earliest cellular adaptations to stress. Neural precursor cells (LUHMES) were differentiated to homogeneous cultures of fully postmitotic human dopaminergic neurons, and then exposed to the mitochondrial respiratory chain inhibitor MPP+ (5 μM). At 18–24 h after treatment, intracellular ATP and mitochondrial integrity were still close to control levels, but pronounced transcriptome and metabolome changes were seen. Data on altered glucose flux, depletion of phosphocreatine and oxidative stress (e.g., methionine sulfoxide formation) confirmed the validity of the approach. New findings were related to nuclear paraspeckle depletion, as well as an early activation of branches of the transsulfuration pathway to increase glutathione. Bioinformatic analysis of our data identified the transcription factor ATF-4 as an upstream regulator of early responses. Findings on this signaling pathway and on adaptive increases of glutathione production were confirmed biochemically. Metabolic and transcriptional profiling contributed complementary information on multiple primary and secondary changes that contribute to the cellular response to MPP+. Thus, combined ‘Omics' analysis is a new unbiased approach to unravel earliest metabolic changes, whose balance decides on the final cell fate.

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“…Transfection of differentiated Luhmes cells has been described previously (Schildknecht, et al, 2013). Briefly, cells were pre-differentiated for 2 days.…”

Section: Methodsmentioning

confidence: 99%

Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction

Ming

Moussaud

Jiang

et al. 2015

Neurobiology of Aging

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The pathological hallmark of Parkinson’s disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation/aggregation. Using two neuronal cell models we found that extracellular ATP induced a significant increase in intracellular αsyn levels between 24 to 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells’ response to extracellular ATP. Although Ca2+ influx via P2X1 receptor is necessary for αsyn accumulation, Ca2+ influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD.

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Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases

Cited by 66 publications

References 58 publications

Cell-based assays for Parkinson's disease using differentiated human LUHMES cells

Cell-based assays for Parkinson's disease using differentiated human LUHMES cells

Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP+

Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction

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