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TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis

1
UMR 1253, iBrain, University of Tours, Inserm, 37000 Tours, France
2
CNRS ERL7001, EA 7501 GICC, University of Tours, 37000 Tours, France
3
CHU de Tours, Service de Biochimie et Biologie Moléculaire, 37000 Tours, France
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Cells 2020, 9(1), 68; https://doi.org/10.3390/cells9010068
Received: 1 October 2019 / Revised: 19 December 2019 / Accepted: 23 December 2019 / Published: 26 December 2019
Cytoplasmic TDP-43 aggregates are a hallmark of amyotrophic lateral sclerosis (ALS). Today, only two drugs are available for ALS treatment, and their modest effect prompts researchers to search for new therapeutic options. TDP-43 represents one of the most promising targets for therapeutic intervention, but reliable and reproducible in vitro protocols for TDP-43-mediated toxicity are lacking. Here, we used HEK293T cells transfected with increasing concentrations of TDP-43-expressing plasmid to evaluate different parameters of toxicity and alterations in cellular metabolism. Overexpression of TDP-43 induced aggregates occurrence followed by the detection of 25- and 35-kDa forms of TDP-43. TDP-43 overexpression decreased cell viability and increased cells arrested at G2/M phase and nuclear fragmentation. Analysis of the energetic metabolism showed a tendency to decrease oxidative phosphorylation and increase glycolysis, but no statistical differences were observed. Metabolomics revealed alterations in different metabolites (mainly sphingolipids and glycerophospholipids) in cells overexpressing TDP-43. Our data reveal the main role of TDP-43 aggregation in cellular death and highlight novel insight into the mechanism of cellular toxicity induced by TDP-43. Here, we provide a simple, sensitive, and reliable protocol in a human-derived cell line to be used in high-throughput screenings of potential therapeutic molecules for ALS treatment. View Full-Text
Keywords: ALS; TDP-43; aggregation; cellular death; apoptosis; metabolomics ALS; TDP-43; aggregation; cellular death; apoptosis; metabolomics
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Lanznaster, D.; Bourgeais, J.; Bruno, C.; Hergesheimer, R.C.; Thepault, R.-A.; Vourc’h, P.; Corcia, P.; Andres, C.R.; Herault, O.; Blasco, H. TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis. Cells 2020, 9, 68.

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