Journal
ALTEX-ALTERNATIVES TO ANIMAL EXPERIMENTATION
Volume 30, Issue 4, Pages 427-444Publisher
SPEKTRUM AKADEMISCHER VERLAG-SPRINGER-VERLAG GMBH
DOI: 10.14573/altex.2013.4.427
Keywords
LUHMES; dopaminergic neurons; alpha-synuclein; neurotoxicity
Categories
Funding
- Doerenkamp-Zbinden Foundation
- Collaborative Research Center 969 Chemical and Biological Principles of Cellular Proteostasis
- Deutsche Forschungsgemeinschaft (DFG)
Ask authors/readers for more resources
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
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available