All plasmids were purchased from and customized by Origene. cDNA sequences were based on the accession number NM_007375.3 for human TARDBP. The WT construct was cloned into the pCMV6-AN-myc vector for expression of N-terminally tagged WT or mutant TDP-43 protein. Custom mutagenesis to generate the A90V (point mutation c.269C>T) and M337V (point mutation c.1009A>G) TDP-43 amino acid substitutions were carried out by Origene. The presence of each mutation was confirmed by DNA sequencing performed by GENEWIZ (forward sequencing primer 5′-GGACTTTCCAAAATGTCG-3′; reverse sequencing primer 5′-ATTAGGACAAGGCTGGTGGG-3′).

SH-SY5Y cells were grown at 37°C with 5% CO₂ in a humidified atmosphere in minimum essential medium (MEM; Thermo Fisher Scientific) supplemented with L-glutamine, 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were subcultured every 3–4 days. For immunofluorescence analysis, cells were grown on glass coverslips coated with 1 mg/mL poly-L-lysine (Sigma) in 24-well plates (Cellstar). For Western blot analysis, cells were grown in 6-well plates (Costar). Cells were transiently transfected using Fugene HD (Promega) following the manufacturer’s instructions 24 h after seeding (Fugene HD:DNA ratio 3:1). Total transfection time was 48 h.

The following antibodies were used for immunofluorescence staining: rabbit N-terminal TDP-43 (1:500; 10782-2-AP, Proteintech), mouse anti-myc 9E10 (1:200; Developmental Studies Hybridoma Bank, 1:200: sc-40, Santa Cruz), mouse anti-scaffold attachment factor B (SAFB) 6F7 (1:300; ab8060, Abcam), mouse anti-protein-associated splicing factor (PSF) (1:50; P2860, Sigma), mouse anti-ubiquitin P4D1 (1:50; sc-8017, Santa Cruz). Anti-mouse and anti-rabbit secondary antibodies coupled to Alexa-488, Alexa-555, and Alexa-568 were used for detection (1:300; Thermo Fisher Scientific). For Western blot analysis, the following antibodies were used: rabbit C- and N-terminal TDP-43 (1:2,500; 12892-1-AP, 1:5,000; 10782-2-AP, Proteintech), mouse S409/410 phospho-TDP-43 (1:2,000; CAC-TIP-PTD-M01, Cosmo), mouse α-tubulin (1:15,000; ab80779, Abcam), rabbit HDAC6 (1:1,000; 12834-1-AP, Proteintech), mouse G3BP (1:1,000; 611126, BD Biosciences), mouse acetylated α-tubulin (1:3,000; 12152, Cell Signaling Technologies), mouse GAPDH 6C5 (1:2,000: sc-32233, Santa Cruz). Anti-mouse and anti-rabbit horseradish peroxidase-coupled secondary antibodies were purchased from Jackson Immunoresearch (1:7,000).

Cells were washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (in PBS) for 15 min followed by permeabilization in 0.25% Triton-X (in PBS) for 10 min. Ten percent normal goat serum (in PBS; Abcam) was used as a blocking reagent. Cells were blocked for 1 h at room temperature and incubated overnight at 4°C in primary antibody solution. The next day, cells were washed with PBS and incubated for 1 h in secondary antibody solution. Coverslips were mounted with Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Image acquisition was performed using a Nikon A1 confocal/Eclipse Ti inverted microscope system and NIS Elements software (Nikon).

The extraction protocol was adapted from Cohen et al. (2012). Cells were grown in 6-well plates and transfected 24 h after seeding. After 48 h, transfected cells were lysed in 300 μL radio-immunoprecipitation assay (RIPA) buffer supplemented with 2 mM EDTA, 1 mM EGTA, protease inhibitors (Complete, Roche), and phosphatase inhibitors (PhosStop, Roche). Lysates were sonicated 2x 15 s with 20% maximum amplitude and centrifuged for 30 min at 100,000 × g and 4°C (Beckman Optima TLX ultracentrifuge with TLA100.3 rotor and Delrin adaptors). The supernatant was collected as the RIPA-soluble fraction. The pellet was washed in RIPA buffer and centrifuged for an additional 30 min at 100,000 × g and 4°C. The supernatant was discarded and the pellet was re-extracted in 100 μL urea buffer [7 M urea, 2 M thiourea, 30 mM Tris pH 8.5, 4% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, Sigma)]. The samples were sonicated 2x 15 s and centrifuged at room temperature for 30 min at 100,000 × g. The supernatant was collected as the urea-soluble fraction.

Cells were lysed in RIPA buffer supplemented with 2 mM EDTA, 1 mM EGTA, protease inhibitors (Complete, Roche) and phosphatase inhibitors (PhosStop, Roche), and the protein concentration was determined by bicinchoninic acid (BCA) assay. RIPA and urea fractions obtained from sequential extractions were diluted 1:1 with RIPA buffer. Proteins were separated using standard Tris-glycine SDS-PAGE gels (polyacrylamide concentrations 8% for HDAC6, all others 10%) and transferred onto nitrocellulose (BioRad) or polyvinylidene difluoride (PVDF; Millipore) membranes. All blocking and antibody incubation steps were performed either in 5% milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 (TBS-T) or in 3% bovine serum albumin (BSA) in TBS-T. Western blots were developed with enhanced chemiluminescent substrates (ECL). Digital images were acquired with a ChemiDoc MP imaging system (BioRad) and quantified with Image Lab 5 (BioRad). Where necessary, blots were stripped with stripping buffer for 15 min (Restore, Thermo Fisher Scientific) and reprobed with loading control antibodies.

The composite schematic of the full structure of human TDP-43 was produced from the nuclear magnetic resonance (NMR) structures 2N4P (Mompean et al., 2015, 2016), 2CQG (Suzuki et al., 2005), 1WF0 (He et al., 2004), and 2N2C (Lim and Song, 2015; Lim et al., 2016) [obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB; Berman et al., 2000)] via iterative homology modeling and loop building using the Molecular Operating Environment (MOE) software (Molecular Operating Environment [MOE], 2017).

All data were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 6 software (San Diego, CA, USA). Normality was established using the Shapiro–Wilk test. Mean differences between groups were analyzed by one-way ANOVA and Tukey’s multiple comparisons test. The threshold for statistical significance was p ≤ 0.05. Experiments were replicated a minimum of three times.

Nuclear magnetic resonance and X-ray structural analysis of individual domains of WT and mutant TDP-43 continue to reveal insights into the relationships between disease-related mutations and the biophysical stability of the protein. We reasoned that assembling a three-dimensional model of TDP-43 may help us predict the structural impact of the A90V variant and support our in vitro efforts in elucidating its effects. Thus, we attempted to collate all structural information obtained from NMR and X-ray crystallography available in the public domain and obtain a complete 3D model of TDP-43 (Figure 1). However, structures including all domains which might enable a comprehensive examination of the effects of specific mutations on overall structure and stability are challenging and not yet realized. The human TDP-43 sequence (Figure 1A) and NMR structures (Figure 1B) in Figure 1 highlight the NLS, NES, RRM1, RRM2, glycine-rich domain, as well as the A90V and M337V mutation sites and the S409/S410 phosphorylation site (He et al., 2004; Suzuki et al., 2005; Kuo et al., 2014; Lim and Song, 2015; Mompean et al., 2015, 2016; Chiang et al., 2016; Lim et al., 2016). The piecewise solutions of NMR and X-ray structures provide valuable domain-level information, but key portions of the protein remain either unresolved or missing. Iterative homology modeling and loop building techniques can be used to infer composite structures from their individual domains. However, in the case of TDP-43 a suitable template structure spanning across multiple domains does not exist, prohibiting the ability to confidently assess the overall structure (only an approximate schematic of the overall structure is shown, Figure 1C) and thus the impact of the A90V and M337V amino acid substitutions. Without a priori assumptions of the impact of the A90V variant based on a complete model, we then proceeded to investigate the consequences of expressing the A90V variant on common pathological phenotypes associated with disease: cytoplasmic relocalization, and the formation of insoluble TDP-43 and TDP-43 protein fragments. We contrasted the A90V variant with the well-characterized mutant M337V, which causes autosomal-dominant familial ALS (Sreedharan et al., 2008).

According to Winton et al. (2008b), transient expression of the A90V variant of TDP-43 in QBI-293 cells results in the aberrant relocalization from the nucleus to the cytoplasm. We first verified expression of N-terminally myc-tagged TDP-43 constructs in SH-SY5Y cells by Western blot using an N-terminal TDP-43 antibody (Figure 2A). Steady state levels of total TDP-43 protein were significantly increased in cells expressing M337V mutant TDP-43 compared to WT expressing cells (0.46 ± 0.19-fold increase over WT; Tukey’s multiple comparisons analysis; p = 0.04), whereas there was no significant difference in cells expressing the A90V TDP-43 variant compared to WT protein (0.17 ± 0.09-fold over WT; Tukey’s multiple comparisons analysis; p = 0.57; Figure 2B; one-way ANOVA, F_2,15 = 3.757; P = 0.0316). We confirmed that this increase in M337V TDP-43 protein levels was not caused by increased levels of mRNA, which were comparable between genotypes (Supplementary Figure S1).

We then performed immunofluorescence staining of transiently transfected cells in order to confirm the relocalization phenotype observed by Winton et al. (2008b) (Figures 2C,D). In agreement with the previous report (Winton et al., 2008b), we observed a significant increase in the proportion of cells with cytoplasmic TDP-43 localization in A90V, compared to WT TDP-43 expressing cells (WT: 28.4 ± 3.0% of cells; A90V: 41.7 ± 3.7% of cells; Tukey’s multiple comparisons analysis; p = 0.041). Interestingly, the proportion of transfected cells with cytoplasmic TDP-43 localization with A90V was very similar compared to the highly pathogenic mutant M337V (WT: 28.4 ± 3.0% of cells; M337V: 43.0 ± 1.6% of cells; Tukey’s multiple comparisons analysis; p = 0.028; one-way ANOVA F_2,6 = 7.679; P = 0.0222; Figure 2D).

In a small number of transfected cells analyzed by immuno fluorescence staining we detected a punctate staining pattern for TDP-43 in the nucleus (Figure 2E). These TDP-43-positive puncta were only rarely observed in SH-SY5Y cells transfected with WT TDP-43 (0.28 ± 0.10% of transfected cells), but were found in significantly higher numbers in cells expressing the pathogenic mutant M337V (2.00 ± 0.60% of transfected cells; Tukey’s multiple comparisons analysis; p = 0.049; Figure 2F). However, we saw no significant difference in puncta formation between WT and A90V TDP-43 expressing cells (A90V: 1.06 ± 0.44% of transfected cells; Tukey’s multiple comparisons analysis; p = 0.44; one-way ANOVA F_2,9 = 3.94; P = 0.059). In order to more closely investigate the composition of these TDP-43-containing puncta, we performed co-staining experiments with two nuclear markers: the nuclear stress body marker SAFB and the paraspeckle marker PSF (Figure 3). We qualitatively distinguished between transfected cells with small, numerous nuclear TDP-43-positive puncta (Figure 2E, asterisk; Figures 3A–C, lower panel) and cells with few, but large, puncta (Figure 2E, arrowhead; Figures 3A–C, upper panel). We observed no co-localization between TDP-43 protein and either SAFB or PSF in small puncta (Figures 3A,B). In cells harboring large TDP-43-positive puncta, both SAFB and PSF were found to be excluded from these structures. We hypothesized that these puncta represent intranuclear inclusions of insoluble, aggregated TDP-43 protein. As aggregated TDP-43 is found pathologically ubiquitinated (Neumann et al., 2006), we stained myc-TDP-43-transfected cells with an anti-ubiquitin antibody (Figure 3C). As expected, we detected diffuse ubiquitin staining in both the nucleus and cytoplasm. Both small and large TDP-43 nuclear puncta were shown to co-localize with ubiquitin, indicating that they are bona fide aggregates of ubiquitinated TDP-43 protein.

In order to further investigate how the A90V and M337V mutations affect the cell biology of TDP-43, we measured a range of cellular phenotypes which have been implicated in disease pathogenesis. Initially, we looked at the formation of insoluble TDP-43 aggregates. We fractionated lysates of transfected SH-SY5Y cells into RIPA-soluble and RIPA-insoluble, urea-soluble fractions by high speed centrifugation and separated proteins by SDS-PAGE. We used a TDP-43 antibody recognizing the N-terminus of TDP-43 to assess total TDP-43 protein levels in the RIPA and urea fractions (Figure 4A). In line with previous reports (Johnson et al., 2009; Bilican et al., 2012; Janssens et al., 2013), we found that the M337V mutation caused accumulation of insoluble TDP-43 in the urea fraction compared to WT (3.21 ± 1.14-fold increase over WT; Tukey’s multiple comparisons analysis; p = 0.023; one-way ANOVA F_2,9 = 5.38; P = 0.029). While an enrichment of TDP-43 in the urea fraction was also observed in A90V-expressing cells (1.59 ± 0.38-fold increase compared to WT), the increase was not statistically significant (Tukey’s multiple comparisons analysis; p = 0.283; Figure 4B).

We used a phospho-specific TDP-43 antibody raised against the disease-relevant phosphorylation sites at S409/S410 (Neumann et al., 2009) to assess the phosphorylation status of the insoluble TDP-43 protein species (Figure 4A). We normalized the phospho-signal in the urea-soluble fractions to the total TDP-43 detected in these fractions to control for the relative increase in insoluble TDP-43 protein in mutant expressing cells (Figure 4C). Immunoblotting with this antibody revealed significantly increased phosphorylation levels at the S409/S410 residues in the urea-soluble fraction of M337V TDP-43 expressing cells compared to cells expressing WT TDP-43 (6.38 ± 2.56-fold increase compared to WT; Tukey’s multiple comparisons analysis; p = 0.036). We detected a non-statistically significant increase in S409/S410 phosphorylation in cells transfected with the A90V variant (1.35 ± 0.48-fold increase compared to WT; Tukey’s multiple comparisons analysis; p = 0.80; one-way ANOVA F_2,9 = 5.015; P = 0.0344). Taken together, these results indicate that while the A90V and M337V mutations induce similar levels of cytoplasmic relocalization of TDP-43, a pathology commonly associated with disease, the effects of the M337V variant on protein solubility and phosphorylation are far greater than those of the A90V mutation. In fact there looks to be a spectrum of pathology in these transfected cell models with WT and M337V TDP-43 at the opposite extremes, with the A90V TDP-43 somewhere in between, in the absence of any additional stressors.

In addition to abnormal phosphorylation as well as decreased protein solubility and aberrant cellular localization, another pathological feature of TDP-43 is the formation of C-terminal truncated protein fragments (Neumann et al., 2006; Mackenzie et al., 2007; Hasegawa et al., 2008). In order to detect C-terminal protein fragments not picked up by the N-terminal TDP-43 antibody used in Figure 4 we extracted soluble and insoluble TDP-43 from transfected SH-SY5Y cells and used a C-terminal antibody (Figures 5A,B). We identified two smaller protein species present only in TDP-43-transfected cells, but not in the vector control cells (Figure 5B). The larger fragment ran close to the 34 kDa marker, while the smaller fragment ran close to the 26 kDa marker. C-terminal TDP-43 fragments with molecular weights of 35 kDa (CTF35) and 25 kDa (CTF25) have been described in a variety of cellular and animal models (Winton et al., 2008a; Xu et al., 2011; Bilican et al., 2012; Choksi et al., 2014; D’Alton et al., 2014; Li et al., 2015), and we will thus refer to the identified truncated TDP-43 species as CTF35 and CTF25. We quantified the amount of both fragments in the RIPA-soluble and urea-soluble fractions. We normalized to the amount of full-length TDP-43 in each sample in order to account for the increase of total TDP-43 protein. In the RIPA-soluble fraction, we observed an accumulation of CTF35 in M337V-transfected cells compared to either WT or A90V-TDP-43 (2.03 ± 0.44-fold increase over WT, p = 0.005; 0.68 ± 0.25-fold increase over A90V, p = 0.044; Tukey’s multiple comparisons analysis; one-way ANOVA F_2,6 = 13.94; P = 0.0056; Figure 5C). However, we did not detect a significant increase of the smaller CTF25 in mutant-transfected cells (M337V: 1.09 ± 0.6-fold increase over WT, p = 0.19; 0.64 ± 0.52-fold increase over A90V, p = 0.36; Tukey’s multiple comparisons analysis; one-way ANOVA F_2,6 = 2.166; P = 0.196). In the urea-soluble fractions the effect of the M337V mutant on the formation of these C-terminal fragments was even more striking: we detected a 20.0 ± 6.6-fold increase in the 25 kDa fragment in the M337V mutant compared to WT (Tukey’s multiple comparisons analysis; p = 0.026) and a 4.3 ± 1.7-fold increase compared to the A90V variant (Tukey’s multiple comparisons analysis; p = 0.049; one-way ANOVA F_2,6 = 7.657; P = 0.0223; Figure 5D). We could not detect any 35 kDa fragment in the urea fraction of WT-TDP-43 transfected cells and found a 5.4 ± 1.1-fold increase in CTF35 levels in M337V- compared to A90V-transfected cells (M337V compared to WT: p = 0.0008: compared to A90V: p = 0.0019; Tukey’s multiple comparisons analysis; one-way ANOVA F_2,6 = 31.53; P = 0.0007).

We hypothesized that the M337V mutant of TDP-43 not only impacts TDP-43 localization and post-translational modifications in a manner more detrimental than the A90V variant, but that the downstream functional consequences of expressing M337V TDP-43 might reflect this more severe phenotype. Such a functional consequence could be altered regulation of transcriptional targets of TDP-43. Previous studies have shown that both depletion of endogenous TDP-43 as well as overexpression of R361S mutant TDP-43 lead to a decrease in G3BP, a regulator of stress granule assembly (McDonald et al., 2011; Aulas et al., 2012). We investigated expression levels of G3BP following 48 h of TDP-43 overexpression and found that only M337V, but not A90V, mutant TDP-43 significantly downregulated G3BP compared to WT TDP-43 and mock control (25.8 ± 8.3% compared to WT, p = 0.01; 23.0 ± 7.8% compared to mock, p = 0.03; Tukey’s multiple comparisons analysis; one-way ANOVA F_3,20 = 4.861; P = 0.0106; Figures 6A,C). Furthermore, levels of HDAC6, a known target of TDP-43 that is likewise downregulated upon TDP-43 silencing (Fiesel et al., 2010), were significantly reduced in M337V TDP-43 expressing cells compared to cells transfected with WT and A90V TDP-43 or mock-transfected controls (WT: 36.1 ± 13.6%; p = 0.017; A90V: 35.6 ± 12.3%; p = 0.02; mock: 43.0 ± 10.2%; p = 0.002; Tukey’s multiple comparisons analysis; one-way ANOVA F_3,12 = 8.455; P = 0.0027; Figures 6B,D). We also probed for levels of acetylated α-tubulin, a substrate for HDAC6 (Hubbert et al., 2002), but found no significant increase in acetylated α-tubulin levels in M337V-transfected cells compared to WT (p = 0.99) or A90V (p = 0.99) TDP-43 (Tukey’s multiple comparisons analysis; Figures 6B,E), suggesting that the decrease in HDAC6 protein levels in M337V-expressing cells is not sufficient to impair acetylation of α-tubulin. In comparison to mock-transfected controls, both WT and A90V-transfected cells showed a small, but significant upregulation of acetylated α-tubulin (WT: 15.8 ± 4.5%, p = 0.05; A90V: 16.0 ± 3.8%, p = 0.04; Tukey’s multiple comparisons analysis) and M337V-transfected cells showed a similar trend (14.2 ± 5.2%, p = 0.08; one-way ANOVA F_3,20 = 3.833; P = 0.0256). An increase in α-tubulin acetylation has been described in conditions of cellular stress (Mackeh et al., 2014), which could be caused by the overexpression of TDP-43 irrespective of mutation. In conclusion, the M337V variant of TDP-43, but not the A90V variant, impacted the expression of known targets of TDP-43 in a manner consistent with the effects of a knockdown of TDP-43 function.