A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long-term exposure and cellular resilience analysis
| Autor: | Johannes Delp, Helena T. Hogberg, Thomas Hartung, Georgina Harris, Lena Smirnova, Marcel Leist, David Pamies, Marize Campos Valadares, Tanja Waldmann |
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| Jazyk: | angličtina |
| Předmět: |
0301 basic medicine
Cellular differentiation Health Toxicology and Mutagenesis Cell Culture Techniques Drug Evaluation Preclinical Drug Resistance Transsulfuration Toxicology 3D culture Neurotoxicity Resiliencemicro RNA Rotenone Antiparkinson Agents chemistry.chemical_compound Toxicity Tests Chronic Cell Aggregation Cell Line Transformed Regulation of gene expression Microscopy Confocal microRNA Dopaminergic Cell Differentiation General Medicine Cell aggregation Recombinant Proteins 3. Good health Cell biology In Vitro Systems Neuroprotective Agents 3D culture Cell Survival Neurotoxins Nanotechnology Nerve Tissue Proteins Biology 03 medical and health sciences Imaging Three-Dimensional ddc:570 Rotenone medicine Neurites Toxicity Tests Acute Neurotoxicity Humans Resilience Dopaminergic Neurons medicine.disease Luminescent Proteins 030104 developmental biology chemistry Gene Expression Regulation Microscopy Fluorescence Cell culture |
| Zdroj: | Europe PubMed Central Archives of Toxicology |
| ISSN: | 0340-5761 |
| DOI: | 10.1007/s00204-015-1637-z |
| Popis: | Several shortcomings of current Parkinson’s disease (PD) models limit progress in identification of environmental contributions to disease pathogenesis. The conditionally immortalized cell line LUHMES promises to make human dopaminergic neuronal cultures more easily available, but these cells are difficult to culture for extended periods of time. We overcame this problem by culturing them in 3D with minor medium modifications. The 3D neuronal aggregates allowed penetration by small molecules and sufficient oxygen and nutrient supply for survival of the innermost cells. Using confocal microscopy, gene expression, and flow cytometry, we characterized the 3D model and observed a highly reproducible differentiation process. Visualization and quantification of neurites in aggregates was achieved by adding 2 % red fluorescent protein-transfected LUHMES cells. The mitochondrial toxicants and established experimental PD agents, rotenone and MPP+, perturbed genes involved in one-carbon metabolism and transsulfuration pathways (ASS1, CTH, and SHTM2) as in 2D cultures. We showed, for the first time in LUHMES, down-regulation of mir-7, a miRNA known to target alpha-synuclein and to be involved in PD. This was observed as early as 12 h after rotenone exposure, when pro-apoptotic mir-16 and rotenone-sensitive mir-210 were not yet significantly perturbed. Finally, washout experiments demonstrated that withdrawal of rotenone led to counter-regulation of mir-7 and ASS1, CTH, and SHTM2 genes. This suggests a possible role of these genes in direct cellular response to the toxicant, and the model appears to be suitable to address the processes of resilience and recovery in neurotoxicology and Parkinson’s disease in future studies. Electronic supplementary material The online version of this article (doi:10.1007/s00204-015-1637-z) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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