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

Lanznaster, Débora; Bourgeais, Jérôme; Bruno, Clément; Hergesheimer, Rudolf; Thépault, Rose-Anne; Vourc’h, Patrick; Corcia, Philippe; Andrés, Christian; Hérault, Olivier; Blasco, Hélène

doi:10.3390/cells9010068

Cited by 9 publications

(12 citation statements)

References 38 publications

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“…in human cells, as already reported (Lanznaster et al, 2019). While the NCL-GFP construct by itself did not affect cell viability, it strikingly antagonized the deadly effects of both WT and mutant TDP-43 in co-transfected cells (Figure 8A), indicating that NCL exerts a protective effect against TDP-43 toxicity also in mammalian cells.…”

Section: Ncl Antagonizes Tdp-43 Cytotoxicity and Aggregation In Human Cells Promoting Tdp-43 Nuclear Localizationsupporting

confidence: 83%

Nucleolin Rescues TDP-43 Toxicity in Yeast and Human Cell Models

Peggion

Massimino

Stella

et al. 2021

Front. Cell. Neurosci.

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TDP-43 is a nuclear protein involved in pivotal processes, extensively studied for its implication in neurodegenerative disorders. TDP-43 cytosolic inclusions are a common neuropathologic hallmark in amyotrophic lateral sclerosis (ALS) and related diseases, and it is now established that TDP-43 misfolding and aggregation play a key role in their etiopathology. TDP-43 neurotoxic mechanisms are not yet clarified, but the identification of proteins able to modulate TDP-43-mediated damage may be promising therapeutic targets for TDP-43 proteinopathies. Here we show by the use of refined yeast models that the nucleolar protein nucleolin (NCL) acts as a potent suppressor of TDP-43 toxicity, restoring cell viability. We provide evidence that NCL co-expression is able to alleviate TDP-43-induced damage also in human cells, further supporting its beneficial effects in a more consistent pathophysiological context. Presented data suggest that NCL could promote TDP-43 nuclear retention, reducing the formation of toxic cytosolic TDP-43 inclusions.

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Section: Ncl Antagonizes Tdp-43 Cytotoxicity and Aggregation In Human Cells Promoting Tdp-43 Nuclear Localizationsupporting

confidence: 83%

Nucleolin Rescues TDP-43 Toxicity in Yeast and Human Cell Models

Peggion

Massimino

Stella

et al. 2021

Front. Cell. Neurosci.

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“…(either WT or bearing the Q331K missense mutation) chimera were signi cantly less viable (around 50%) compared to control cells (i.e., cells co-transfected with plasmids encoding GFP and mKate-2 only, Fig. 9A), highlighting the cytotoxic potential of TDP-43 overexpression in human cells, as already reported [69]. Co-expression of the NCL-GFP construct strikingly antagonized the deadly effects of both WT and mutant TDP-43 ( Fig.…”

Section: ) Ncl Antagonizes Tdp-43 Cytotoxicity and Aggregation In Husupporting

confidence: 74%

Nucleolin rescues TDP-43 toxicity in yeast and human cell models

Peggion

Massimino

Stella

et al. 2020

Preprint

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Background TDP-43 is a nuclear protein involved in pivotal processes, extensively studied for its implication in neurodegenerative disorders. TDP-43 cytosolic inclusions are a common neuropathologic hallmark in amyotrophic lateral sclerosis (ALS) and related diseases, and it is now established that TDP-43 misfolding and aggregation play a key role in their etiopathology. TDP-43 neurotoxic mechanisms are not yet clarified, but the identification of proteins able to modulate TDP-43-mediated damage may provide crucial information to unveil the molecular basis of TDP-43 proteinopathies. Methods Here we generated and characterized novel models of TDP-43 toxicity in the yeast S. cerevisiae, which were used to investigate the effect of the nucleolar protein nucleolin (NCL) on TDP-43-damaged yeast cells, by employing multiple approaches (genetics, biochemistry, microscopy). We further characterized the NCL-TDP-43 relationship in human HEK293T cells, by the combination of biochemical and microscopy-based assays. Results We show for the first time that NCL acts as a potent suppressor of TDP-43 toxicity in yeast models, since NCL overexpression is able to rescue TDP-43-dependent damage on cell viability and morphology, by reducing the levels of TDP-43 aggregates, thus proteostatic stress. Interestingly, data in yeast cells point to the implication of the extra-nuclear fraction of NCL in the suppressive effect. We further provide evidence that NCL co-expression alleviates the TDP-43-induced toxicity also in HEK293T cells, as indicated by the restoration of cell viability, and the diminished apoptosis activation. Importantly, biochemical and microscopy data indicate that NCL protein in human cells reduces the amount of TDP-43 inclusions. Collectively, results in HEK293T cells further support the beneficial effects of NCL on TDP-43-dependent toxicity in a more consistent pathophysiological context. Conclusions Altogether, data in yeast and human cell models demonstrate that NCL potently supresses the cytotoxicity caused by the TDP-43 protein, and further suggest that NCL could act by promoting the TDP-43 nuclear retention, and thus reducing the formation of cytosolic TDP-43 toxic aggregates. Pinpointing NCL as a novel player in mediating TDP-43 toxicity, experimental evidence could support NCL as promising therapeutic target in ALS and ALS-related disorders.

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“…HEK-293T cells (American Type Culture Collection, Manassas, VA, USA) were the cell line of choice due to its robust transfection efficiency and common application in studies on TDP-43 proteinopathy [ 11 , 13 , 16 , 18 ]. We maintained cells in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% ( v / v ) fetal bovine serum (FBS) at 37 °C (Gibco, Strasbourg, France) and in an incubator maintaining an atmosphere of 5% CO 2 (Invitrogen, Strasbourg, France).…”

Section: Methodsmentioning

confidence: 99%

“…Interestingly TDP-43 has been indirectly linked to metabolic dysfunction. For example, studies have highlighted perturbations in the carnitine shuttle and fatty acid oxidation in mitochondria of transgenic Drosophila overexpressing TDP-43 [ 15 ], as well as in glycerophospholipid metabolism in a HEK-293T (Human Embryonic Kidney 293T) model [ 16 ]. Accordingly, one could suggest an effect of TDP-43 propagation on cellular metabolism, which may be associated with its toxicity.…”

Section: Introductionmentioning

confidence: 99%

Conditioned Medium from Cells Overexpressing TDP-43 Alters the Metabolome of Recipient Cells

Hergesheimer

Lanznaster

Bourgeais

et al. 2020

Cells

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive death of both upper and lower motor neurons. The disease presents a poor prognosis, and patients usually die 2–5 years after the onset of symptoms. The hallmark of this disease is the presence of phosphorylated and ubiquitinated aggregates containing trans-active response DNA-binding protein-43 (TDP-43) in the cytoplasm of motor neurons. TDP-43 pathology has been associated with multiple pathways in ALS, such as metabolic dysfunction found in patients and in in vivo models. Recently, it has been described as a “prion-like” protein, as studies have shown its propagation in cell culture from ALS brain extract or overexpressed TDP-43 in co-culture and conditioned medium, resulting in cytotoxicity. However, the cellular alterations that are associated with this cytotoxicity require further investigation. Here, we investigated the effects of conditioned medium from HEK293T (Human Embryonic Kidney 293T) cells overexpressing TDP-43 on cellular morphology, proliferation, death, and metabolism. Although we did not find evidence of TDP-43 propagation, we observed a toxicity of TDP-43-conditioned medium and altered metabolism. These results, therefore, suggest (1) that cells overexpressing TDP-43 produce an extracellular environment that can perturb other cells and (2) that TDP-43 propagation alone may not be the only potentially cytotoxic cell-to-cell mechanism.

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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

Cited by 9 publications

References 38 publications

Nucleolin Rescues TDP-43 Toxicity in Yeast and Human Cell Models

Nucleolin Rescues TDP-43 Toxicity in Yeast and Human Cell Models

Nucleolin rescues TDP-43 toxicity in yeast and human cell models

Conditioned Medium from Cells Overexpressing TDP-43 Alters the Metabolome of Recipient Cells

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