Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by the rapid deterioration of the superior and inferior motor neurons, leading to death of the patient 2–5 years following symptom onset [1
]. To this day, no effective treatment exists despite numerous clinical trials. Hence, the continued study of ALS pathogenesis remains critical to better understand the relationship between its pathological mechanisms and motor neuron degeneration. The hallmark of ALS is the presence of phosphorylated and ubiquitinated aggregates containing full-length and cleaved forms of trans-active response DNA-binding protein-43 (TDP-43) in motor neurons [2
]. The toxicity of this aggregation has been a long-debated subject, but there is increasing evidence arguing that it is tightly related to the neurodegeneration observed in ALS cases [4
The C-terminus of TDP-43 has been associated with its propensity to aggregate. This region also exhibits a prion-like structure; it is a low-complexity domain, rich in glycine, asparagine, and glutamine residues, and has an undefined tertiary structure. Prions are classified as abnormally folded, transmissible proteins that seed the misfolding of otherwise normally folded copies of itself, usually causing their aggregation and cytotoxicity [5
]. In cell culture, “prion-like” seeding and propagation of aggregation have been observed for proteins that are heavily involved in neurodegenerative diseases, such as Parkinson’s, Huntington’s and ALS [6
]. More specifically, researchers have observed this “prion-like” behavior in TDP-43 extracted from diseased brains of ALS and frontotemporal lobar dementia (FTLD) patients [9
], as well as from overexpressed TDP-43 in co-culture [12
] and in conditioned medium [13
]. This propagation has been associated with cytotoxicity. However, the cellular alterations related to this toxicity have not been fully investigated.
Increasing evidence supports the hypothesis that metabolism is heavily modified in ALS, with reports on hypermetabolism, altered lipid/glucose metabolism, tricarboxylic acid (TCA) cycle, and mitochondrial function [14
] in ALS patients. 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.
To investigate whether TDP-43 “prion-like” behavior was involved with metabolic disturbances, we performed metabolomics on HEK-293T cells cultured in conditioned medium from other HEK-293T cells having overexpressed TDP-43. Briefly, we overexpressed wild-type (WT) TDP-43 and added the corresponding conditioned medium to naïve recipient HEK-293T cells (Figure 1
). Although we did not observe signs of TDP-43 propagation, the naïve cells exhibited tendencies toward lower structural integrity and higher membrane permeability. In addition, these cells demonstrated a metabolome profile that was different from that of untreated cells and cells overexpressing TDP-43, thus suggesting a defect in whole-cell metabolism. These results, altogether, led us to hypothesize that TDP-43-conditioned medium is associated with cellular demise. Furthermore, the molecular environment within TDP-43-conditioned medium is of utmost importance for the understanding of the relationship among TDP-43 propagation, the extracellular environment, and cytotoxicity.
2. Materials and Methods
The full-length wild-type human TDP-43 sequence was cloned into the mammalian expression vector pcDNA3.3 (Invitrogen, Strasbourg, France) using the following primers: forward_5′- TCTGAATATATTCGGGTAACCGAAG-3′ and reverse_5′- CTAGTGGTGATGGTGATGATGAGAACCCCCCATTCCCCAGCCAGAAGACTTAG-3′ (Eurogentec, Angers, France). We were interested in the wild-type form of TDP-43 because this is the predominant form found in ALS patients, as mutated TDP-43 accounts for 5% of cases [17
]. A histidine tag (6×His) was fused to the C-terminus of wilde-type TDP-43 (wtTDP-43-6×His) to distinguish the overexpressed form from the endogenous form.
2.2. Cell Culture and Generation of Conditioned Medium
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
]. We maintained cells in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% (v
) fetal bovine serum (FBS) at 37 °C (Gibco, Strasbourg, France) and in an incubator maintaining an atmosphere of 5% CO2
(Invitrogen, Strasbourg, France). In all, 700,000 cells were seeded in T-25 flasks (Corning, Paris, France). After 24 h, the seeded cells were either transfected with 8 µg of pcDNA3.3-wtTDP43-6×His vector and 16 µL of jetPEI transfection agent (Polyplus-transfection®
SA, Strasbourg, France) or treated only with an equal quantity of jetPEI. After 4 h, the growth media were replaced with 8 mL of fresh media, and overexpression was allowed for 72 h. Conditioned medium from TDP-43-transfected cells was referred to as TDP-43 CM and that of cells only treated with jetPEI referred to as NT CM. After 72 h of overexpression, the conditioned media were collected and centrifuged at 1500× g
for 5 min at room temperature to remove floating dead cells and debris. The media were then added to Amicon Ultra-15 centrifuge tubes (Merck Millipore Ltd., Saint-Quentin-Fallavier, France) and concentrated by centrifugation at 10,000× g
at room temperature until the volume was reduced 10-fold (Vi
= 8.0 mL; Vf
= 0.8 mL), representing a 10-fold concentrated conditioned medium that was then applied to downstream analyses.
2.3. Enzyme-Linked Immunosorbent Assay (ELISA) on Conditioned Media
Nunc MaxiSorp 96-well plates (Invitrogen, Strasbourg, France) were coated overnight at 4 °C with 10 µg/mL of anti-TDP-43 antibody targeting the N-terminus (polyclonal rabbit, ProteinTech, Manchester, UK). After 24 h, the coated wells were rinsed three times with phosphate-buffered saline supplemented with 0.1% Tween-20 (PBST) and one time with PBS. Wells were saturated with 4% (w/v) powdered milk (Régilait©, Saint-Martin-Belle-Roche, France) in PBS (MPBS) for 2 h at room temperature, rinsed with PBST and PBS in the same manner, and incubated with each of the 10-fold concentrated NT CM and TDP-43 CM, and 10 µg/mL of purified GFP-wtTDP-43-6×His (positive control) for 1 h at room temperature. Wells were then rinsed five times with PBST, one time with PBS, and incubated in MPBST with anti-6×His antibody (monoclonal mouse, 1/5000 dilution, ProteinTech, Manchester, UK) for 1 h at room temperature. After rinsing in the same manner, each well was incubated with 100 µL of room-temperature TMB-ELISA solution (Invitrogen, Strasbourg, France) for 5–10 min. Finally, 50 µL of 2 M H2SO4 were added to stop the reaction, and the absorbance was measured at 450 nm.
2.4. Recipient Cell Culture
Recipient cells were either non-transfected or transfected with pcDNA3.3-wtTDP43-6xHis. Each cell type received either NT CM or TDP-43 CM. Non-transfected cells in NT CM (NT CM/NT cells) represented the healthy control. Transfected cells in NT CM (NT CM/TDP-43 cells) represented cells exhibiting TDP-43 proteinopathy in control medium. Non-transfected cells in TDP-43 CM (TDP-43 CM/NT cells) represented cells without TDP-43 proteinopathy in the conditioned medium of cells having endured TDP-43 proteinopathy. Finally, transfected cells in TDP-43 CM (TDP-43 CM/TDP-43 cells) represented cells displaying both TDP-43 proteinopathy and conditioned medium from the same type of cells.
2.5. Cell Death, Morphology, and Proliferation
Recipient HEK-293T cells were seeded in 6-well plates (Corning, Paris, France) at 250,000 cells/well in 2 mL of DMEM with 5% FBS. After 24 h, cells were either treated only with 6 µL of jetPEI/well (NT cells) or transfected with 3 µg of pcDNA3.3-wtTDP-6×His vector and 6 µL of jetPEI/well (TDP-43 cells). After 24 h, 200 µL of medium were removed from each well, and 200 µL of 10-fold concentrated NT CM or TDP-43 CM were added to the corresponding wells to dilute the conditioned media to 1×. The rationale was to introduce recipient cells to a 1× concentration of conditioned medium without depriving them of the basic nutrients provided by fresh culture medium. This would control for non-specific effects related to old, nutrient-deprived medium. Following a 24 h incubation at 37 °C, cells were trypsinized in 0.25% trypsin (Gibco, Strasbourg, France), washed in PBS, and resuspended in PBS/5% FBS. For cell death, propidium iodide (Sigma-Aldrich®, Lyon, France) was added to samples of 250,000 cells at a final concentration of 10 µg/mL in PBS/1% FBS. This molecule is taken up by dead cells because their plasma membrane becomes less intact. After 30 min of incubation at 37 °C with 5% CO2, cells were placed on ice before being analyzed by a Becton Dickinson Accuri™ C6 Plus flow cytometer (BD, Franklin Lakes, NJ, USA).
Morphology was simultaneously evaluated by sorting cells depending on their side scatter (SSC: granularity) and forward scatter (FSC: size). The distribution of cells from the SSC/FSC ratio of NT CM/NT cells was considered the healthy, morphological control. Densities that deviated from this ratio were considered to have aberrant morphologies.
Proliferation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay (Invitrogen, Strasbourg, France). Recipient cells were seeded in 96-well plates at 10,000 cells/well (Corning, Paris, France) coated with 100 µg/mL poly-D-Lysine (Sigma-Aldrich®, Lyon, France) in 200 µL of DMEM with 5% FBS. Replicates of three were seeded per condition. After 24 h, cells were either treated only with 240 nL of jetPEI/well (NT cells) or transfected with 120 ng of pcDNA3.3-wtTDP-6×His vector and 240 nL of jetPEI/well (TDP-43 cells). After a 24 h incubation at 37 °C with 5% CO2, 20 µL of medium were removed from each well, and 20 µL of 10-fold concentrated NT CM or TDP-43 CM were added to the corresponding wells to dilute the conditioned media to 1×. Following the 24 h incubation at 37 °C, the medium was removed and replaced with 0.5 mg/mL of MTT diluted in Hank’s Balanced Salt Solution (HBSS, Gibco, Strasbourg, France). Following 30 min of incubation at 37 °C, the medium was withdrawn, the precipitated formazan was solubilized with 100 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich®, Lyon, France), and proliferation was quantified by spectrophotometry at a wavelength of 570 nm.
2.6. Immuno-Detection of TDP-43 in Recipient Cells
One million recipient cells in each condition were harvested in ice-cold PBS and centrifuged at 900× g. Cell lysis was induced with ice-cold Pierce-RIPA buffer containing protease inhibitor cocktail (Invitrogen, Strasbourg, France) and universal nuclease (Invitrogen, Strasbourg, France) with agitation for 15 min at 4 °C. Protein content of whole-cell lysates was measured by the Lowry method (Bio-Rad, Marnes-la-Coquette, France). Then, 30 µg of protein from each cell lysate were separated in 4–20% SDS-PAGE gel apparatus (Bio-Rad, Marnes-la-Coquette, France) and transferred to PVDF (polyvinylidine fluoride) membranes. After blocking with 5% (w/v) Régilait in Tris-buffered saline supplemented with 0.2% Tween-20 (TBS-T), membranes were incubated for 1 h at room temperature with the following primary antibodies: mouse HRP-conjugated anti-β-actin (1/100,000 dilution, ProteinTech, Manchester, UK) for an internal control, mouse anti-GFP (1/5000 dilution, Sigma-Aldrich®, Lyon, France) for GFP detection of GFP-6xHis, and mouse HRP-conjugated anti-6×His (1/100,000 dilution, ProteinTech) for detection of GFP-6xHis and wtTDP-43-6×His, respectively. For GFP-specific detection, the primary incubation was followed by 1 h incubation with a secondary antibody coupled to horseradish peroxidase (HRP; anti-mouse, 1/5000; Invitrogen, Strasbourg, France). Chemiluminescence was observed by adding Clarity Western ECL substrate to the membrane followed by visualization using Chemidoc apparatus (Bio-Rad, Marnes-la-Coquette, France) after incubation with ECL. Band intensity was measured with Image Lab software v6.1 (Bio-Rad, Marnes-la-Coquette, France).
2.7. Glycolysis and Cellular Respiration of Recipient Cells
Recipient HEK293T cells were seeded in a Seahorse XF96 microplate (Agilent, Paris, France) at 10,000 cells/well in 200 µL of DMEM with 5% FBS. Replicates of three were seeded per condition. After 24 h, cells were either transfected with 120 ng pcDNA3.3-wtTDP43-6×His vector and 240 nL jetPEI (TDP-43 cells) or only supplemented with an equal amount of jetPEI (NT cells). After a 24 h incubation at 37 °C with 5% CO2, 20 µL of medium were removed from each well and 20 µL of 10-fold concentrated NT CM or TDP-43 CM were added to the corresponding wells to dilute the conditioned media to 1×. Following a 24 h incubation, the cells were analyzed by a Seahorse XF analyzer (Agilent, Paris, France) to measure the following metabolic parameters: basal respiration, proton leak, maximal respiration, spare respiratory capacity, non-mitochondrial respiration, ATP production, non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve.
2.8. Metabolomics Analysis of Recipient Cells
A targeted, quantitative approach was employed based on the AbsolutIDQ™ p180 kit (Biocrates, Innsbruck, Austria) using a flow injection analysis and high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS) protocol (Waters, Etten-Leur, The Netherlands). This assay permits the quantification of 188 metabolites [19
]. Recipient HEK293T cells were seeded in 6-well plates at 250,000 cells/well in DMEM with 5% FBS. After 24 h, cells were either treated only with 6 µL of jetPEI/well (NT cells) or transfected with 3 µg of pcDNA3.3-wtTDP-6×His vector and 6 µL of jetPEI/well (TDP-43 cells). After 24 h, 200 µL of medium were removed from each well, and 200 µL of 10-fold concentrated NT CM and TDP-43 CM were added to the corresponding wells to dilute the conditioned media to 1×. Following a 24 h incubation at 37 °C, recipient cells were trypsinized and centrifuged at 900× g
for 5 min at 4 °C. Approximately one million cells were resuspended in 1 mL of ice-cold PBS and centrifuged using the same parameters as above. Cells were dried by removing all of the PBS and then stored at −80 °C. On the day of analysis, cells were thawed and resuspended in ice-cold ethanol/1× PBS at a ratio of 85:15, then subjected to 3 freeze/thaw cycles in liquid nitrogen. Samples were centrifuged at 18,000× g
at 2 °C for 5 min. Samples were loaded onto a filter paper and dried in a stream of nitrogen for derivation with a solution of 5% phenyl isothiocyanate. Afterward, dried residues were extracted with methanol containing 5 mM ammonium acetate. The MetIDQ®
software (Biocrates, Innsbruck, Austria) was used to calculate the concentrations of individual metabolites. Quality controls (QCs) were analyzed regularly on the plate (every 8 samples) to ensure the stability of the mass spectrometer over time.
2.9. Statistical Analyses
For metabolomics analysis, two separate analytical methods were employed to identify discriminant metabolites—multivariate and univariate analyses. These analyses were performed using MetaboAnalyst v4.0 (http://www.metaboanalyst.ca
). The classification method was based on partial least squares discriminant analysis (PLS-DA). Values of variable importance in projection [20
] represent the importance of the metabolite for the PLS-DA models. The score scatter plots show the classified samples, and the loading plot characterizes the relation between the Y and X variables. For each analysis, the standard, default parameters of MetaboAnalyst were applied. The univariate analysis of metabolite levels was based on the fold-change (FC) values and the threshold of significance (p
< 0.1) after a non-parametric Wilcoxon test (volcano plot), while the multivariate analysis of metabolites depended on the accuracy of the variable importance in the projection (VIP). Therefore, the metabolites that had VIP values > 1 of the multivariate analyses and those that were determined by an FC > 2 (for each comparison) with p
< 0.1 of the univariate analyses were retained as the most relevant for further discussion [21
Results are shown as mean ± standard error of the mean (SEM). Cytometry results were analyzed using FlowJo VX software v.10.0 (BD Biosciences, San Jose, CA, USA). When relevant, the Mann–Whitney non-parametric t-test was performed using Prism v.7.0 (GraphPad Software, San Diego, CA, USA). Results were considered significant with p < 0.05.
The findings of this study suggest that the conditioned medium resulting from cells overexpressing TDP-43 provokes deleterious effects in other cells. These effects seem related to the changes in the metabolome profile of these cells, which could represent the underlying toxic mechanisms. On the one hand, the common metabolome alterations between TDP-43-overexpression and TDP-43-conditioned medium could represent mechanisms that would occur in both cells overexpressing TDP-43 and naïve cells in contact with the associated conditioned medium. On the other hand, the modified metabolites only found in naïve cells with TDP-43-conditioned medium probably indicate distinct and deleterious mechanisms. However, the scope of this study did not allow for the determination of the toxic outcome of such metabolic changes. Therefore, it will be important in future studies to investigate more closely these metabolites to elucidate whether specifically altering them will lead to cytotoxicity. Moreover, future endeavors should include neuronal cell types, such as iPSC-derived motor neurons, to validate these findings.
This study also raises the question as to which species in the conditioned medium acted to bring about such changes. The results shed light on the hypothesis that TDP-43 propagation could not be the only threatening factor in the medium surrounding cells. Since many avenues of the metabolome appeared to be altered in recipient cells, it is unlikely that TDP-43 propagation would be responsible for all outcomes. Furthermore, the release of TDP-43 does not exclude the possibility that other yet-to-be identified species were released in parallel. At this stage, it remains unclear whether the additional factors are specifically linked to overexpressed TDP-43 or non-specifically triggered from the resulting cellular stress. In order to better understand the role of TDP-43 propagation in the context of ALS, these other factors should not be overlooked.