title: A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long-term exposure and cellular resilience analysis.
abstract: 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.
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA. lsmirno1@jhu.edu.
authors.forename: L
authors.initials: L
authors.lastname: Smirnova
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA.
authors.forename: G
authors.initials: G
authors.lastname: Harris
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Delp
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA.
authors.forename: M
authors.initials: M
authors.lastname: Valadares
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA.
authors.forename: D
authors.initials: D
authors.lastname: Pamies
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA.
authors.forename: H T
authors.initials: HT
authors.lastname: Hogberg
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Waldmann
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Leist
authors.affiliation: Center for Alternatives to Animal Testing (CAAT), Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA.|Center for Alternatives to Animal Testing (CAAT), Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Hartung
mesh_terms: D000978:Antiparkinson Agents / Q000494:pharmacology*; D002449:Cell Aggregation; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D059290:Dopaminergic Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004351:Drug Resistance; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D021621:Imaging, Three-Dimensional; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009498:Neurotoxins / Q000633:toxicity*; D011994:Recombinant Proteins / Q000378:metabolism; D023382:Toxicity Tests, Acute / Q000379:methods*; D036401:Toxicity Tests, Chronic / Q000379:methods*
chemical_list: D000978:Antiparkinson Agents; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D009498:Neurotoxins; D011994:Recombinant Proteins
keywords: 3D culture; Neurotoxicity; Resilience; Rotenone; microRNA