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

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.

Keywords: ALS; TDP-43; aggregation; apoptosis; cellular death; metabolomics.

Figure 1

HEK293T transfection with increasing quantities of GFP-TDP-43 induces an increase in TDP-43 expression and cytoplasmic aggregation. (A) i Control cells transfected only with GFP (4 µg) show presence of GFP signal in the whole cell. (A) ii–v Increasing concentrations of GFP-TDP-43 showing different cellular distributions (observed in detail in (A) vi; scale bar = 100 µm). Cell nucleus was counterstained with DAPI (2 μg/mL; Sigma–Aldrich). Scales bar = 200 µm for (A) i–v. (B) The increase in expression was followed by an increase in the number of cells presenting cytoplasmic aggregates (n = 2). (C) and (D) Increase in the number of positive cells and median fluorescence signal for GFP analyzed by flow cytometry (n = 5). Nonparametric Kruskal–Wallis test revealed * p < 0.05 for TDP-43 1 μg when compared to GFP+ group (positive control: cells transfected with 4 μg of GFP plasmid).

Figure 2

TDP-43 overexpression increases TDP-43 protein full-length (43kDa) presence in soluble and insoluble fractions of cells lysates (B) together with the detection of 35- (C) and 25-kDa (D) C-terminal fragments (CTFs). Representative Western blots are shown in panel (A). CE: crude extract; S: soluble fraction; P: insoluble fraction. Nonparametric Kruskal–Wallis test revealed * p < 0.05 and # p = 0.053 compared to the respective control groups (CE x CE; S x S; P x P; n = 3–6).

Figure 3

TDP-43 overexpression induces a decrease in cellular viability measured after 48 h of transfection. (A): Number of alive cells analyzed by trypan blue exclusion (n = 3; * p < 0.05). (B) Cell cycle analysis performed by flow cytometry (n = 4–5; * p < 0.05). (C) Percentage of sub-G1 events (fragmented nuclei) analyzed by flow cytometry (n = 4–5; * p < 0.05). (D) Cell viability analyzed by MTT reduction (n = 4; * p < 0.05). (E) LDH released in the medium (n = 4; * p < 0.05). (F) Percentage of positive cells for PI fluorescence (n = 5–6; * p < 0.05). (G) Microscopic analysis of cells transfected with increasing concentrations of TDP-43 and incubated with PI (scale bar = 200 µm).

Figure 4

Analysis of TDP-43 overexpression, oxidative stress, and impairment in mitochondrial membrane potential in HEK293T cells. (A) Median fluorescence of DCF-DA signal from TDP-43-transfected cells in relation to the control. Statistical analysis revealed no differences between groups. (B) Membrane mitochondrial potential analyzed with TMRM probe revealed no alterations induced by TDP-43 overexpression (n = 3–8). (C) Western blot analysis of p38 phosphorylation in whole-cell lysates from TDP-43-overexpressing cells (n = 4–5).

Figure 5

TDP-43 overexpression induces dose-dependent trends of modifications in energy metabolism. (A) Oxygen consumption rate (OCR); (B) Extracellular acidification rate (ECAR). Data are presented as mean ± SEM. Statistical analysis revealed no differences between groups (Kruskal–Wallis nonparametric test; n = 3–4).

Figure 6

(A) Metabolomics analyses reveal a set of distinguishable metabolites altered in TDP-43-overexpressing HEK293T cells. (B,C) VIP metabolites and volcano plot analysis are represented for TDP-43 4 µg. Metabolomics analyses were realized with MetaboAnalyst (http://www.metaboanalyst.ca).

Figure 7

A fast, reproducible and reliable protocol for assessment of TDP-43-associated toxicity. Our protocol reveals the best markers of toxicity associated with TDP-43 overexpression: evaluation of protein aggregation (analyzed by western blot in differential cell lysates), cell viability (by MTT reduction, LDH release, and PI incorporation), and cell cycle assessment (after cell lysis and PI incorporation). These parameters should be employed for the evaluation of possible therapeutic agents against TDP-43-mediated toxicity and possible application as ALS therapeutics.