title: Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases.
abstract: Human differentiated cell types, such as neurons or hepatocytes, are of limited availability, and their use for experiments requiring ectopic gene expression is challenging. Using the human conditionally-immortalized neuronal precursor line LUHMES, we explored whether genetic modification in the proliferating state could be used for experiments in the differentiated post-mitotic neurons. First, alpha-synuclein (ASYN), a gene associated with the pathology of Parkinson's disease, was overexpressed. Increased amounts of the protein were tolerated without change of phenotype, and this approach now allows further studies on protein variants. Knockdown of ASYN attenuated the toxicity of the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP+). Different lentiviral constructs then were tested: cells labeled ubiquitously with green (GFP) or red fluorescent protein (RFP) allowed the quantification of neurite growth and of its disturbance by toxicants; expression of proteins of interest could be targeted to different organelles; production of two different proteins from a single read-through construct was achieved successfully by an expression strategy using a linker peptide between the two proteins, which is cleaved by deubiquitinases; LUHMES, labeled with GFP in the cytosol and RFP in the mitochondria, were used to quantify mitochondrial mobility along the neurites. MPP+ reduced such organelle movement before any other detectable cellular change, and this toxicity was prevented by simultaneous treatment with the antioxidant ascorbic acid. Thus, a strategy has been outlined here to study new functional endpoints, and subtle changes of structure and proteostasis relevant in toxicology and biomedicine in post-mitotic human cells.
authors.affiliation: Doerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine, Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schildknecht
authors.forename: Christiaan
authors.initials: C
authors.lastname: Karreman
authors.forename: Dominik
authors.initials: D
authors.lastname: Pöltl
authors.forename: Liudmila
authors.initials: L
authors.lastname: Efrémova
authors.forename: Cornelius
authors.initials: C
authors.lastname: Kullmann
authors.forename: Simon
authors.initials: S
authors.lastname: Gutbier
authors.forename: Anne
authors.initials: A
authors.lastname: Krug
authors.forename: Diana
authors.initials: D
authors.lastname: Scholz
authors.forename: Hanne R
authors.initials: HR
authors.lastname: Gerding
authors.forename: Marcel
authors.initials: M
authors.lastname: Leist
mesh_terms: D002460:Cell Line; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000502:physiology; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D006801:Humans; D016086:Lentivirus; D019636:Neurodegenerative Diseases / Q000139:chemically induced* / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*