Abstract
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.
Highlights
Transactive Response DNA Binding Protein 43 (TDP-43) is a ubiquitous 43 kDa-protein implicated in multiple steps of transcriptional and post-transcriptional regulation
Cells transfected with increasing concentrations of GFP-TDP-43 plasmid showed different cellular distributions of the fluorescent signal (Figure 1Aii–v): GFP signal was observed in the nucleus, the cytoplasm, or as aggregates
The ratio of oxygen consumption rate (OCR) max and extracellular acidification rate (ECAR) max suggests the occurrence of the Warburg effect, but we found no statistical differences between control and TDP-43-overexpressing cells
Summary
Transactive Response DNA Binding Protein 43 (TDP-43) is a ubiquitous 43 kDa-protein implicated in multiple steps of transcriptional and post-transcriptional regulation. Full-length TDP-43 protein is found in cytoplasmic aggregates in degenerated motor neurons and surrounding glial cells in the central nervous system of patients suffering from amyotrophic lateral sclerosis (ALS). These aggregates are considered the hallmark of ALS. It remains controversial whether TDP-43 aggregation is the principal cause of the motor neuronal degeneration observed in ALS patients, but recent studies shed a light into the key role of TDP-43 aggregates in causing the observed neurodegeneration. TDP-43 aggregates are present in patients with and without any mutations in the TDP-43 encoding gene, TARDPB, and in patients with mutations in other genes involved in ALS pathology, like SOD-1 and C9orf (reviewed by [3])
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
