Fig 2: NDD-TDP-43 mutations induce phosphorylation, insolubilization, and coaggregation with WT TDP-43.(A) Representative images of Neuro2a cells transiently expressing TDP-43 WT, NDD mutants, or NLS mutant immunostained with anti-FLAG and anti–pTDP-43. (B) Quantification of the percentage of the pTDP-43–positive cells per FLAG-positive cells in (A). Over 100 cells were quantified per experiment. (C) Representative immunoblots of Neuro2a cells transiently transfected with TDP-43-3×FLAG WT and mutants. (D) Quantification of pTDP-43/FLAG ratios (relative to TDP-43WT) in (C). (E) Representative immunoblots of Neuro2a cells transiently expressing TDP-43-3×FLAG WT and mutants. Proteins sequentially extracted with tris-HCl (TS), 1% Triton X-100 (TX), and 1% sarkosyl (Sar), as well as a sarkosyl-insoluble fraction (ppt), were analyzed by immunoblotting. (F) Quantification of the percentage of each fraction relative to the total amount of TDP-43-3×FLAG in (E). (G) Representative immunoblots of the sequentially extracted lysates from Neuro2a cells transiently expressing TDP-436M-3×FLAG full-length (FL) and deletion mutants. (H) Quantification of the percentage of each fraction to the total amount of TDP-43-3×FLAG in (G). (I) Representative immunofluorescence images of Neuro2a cells transiently expressing TDP-436M-3×FLAG (top), TDP-43WT-HA (middle), or both (bottom). Insets show magnified images of the outlined square area. (J) Line scan analysis of coaggregation of TDP-436M-3×FLAG and TDP-43WT-HA in the inset from (I). (K) Representative immunoblots of the sequentially extracted lysates from Neuro2a cells transiently coexpressing TDP-43WT-3×FLAG or TDP-436M-3×FLAG and TDP-43WT-HA. (L) Quantification of the percentage of TDP-43-3×FLAG in each fraction relative to the total amount of TDP-43WT-HA in (K). Scale bars, 10 μm (A and I). n = 3, biologically independent experiments (B, D, F, H, and L). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 [ANOVA with Tukey’s test in (B), (D), (F), and (H) and unpaired two-sided t test in (L)].

Fig 3: Schematic illustration of the role of the TDP-43 monomer-dimer/multimer equilibrium in the development of TDP-43 pathology in ALS.TDP-43 exists in a monomer-dimer/multimer equilibrium in cells. The NTD of TDP-43 mediates the formation of physiological homodimers, which are essential for its physiological splicing function. The RNA export factor Nxf1 facilitates the nuclear export of monomeric TDP-43 (left). Various types of stresses, including transcription inhibition, impair TDP-43 dimerization/multimerization, likely by inducing spliceosomal defects and disrupting the monomer-dimer/multimer equilibrium, resulting in increased amounts of monomeric nonfunctional TDP-43 (middle). This excess of monomeric TDP-43 is exported from the nucleus in an Nxf1-dependent manner, leading to the cytoplasmic mislocalization of TDP-43 and its subsequent phosphorylation and aggregation, resulting in irreversible TDP-43 pathology in ALS (right).

Fig 4: NDD-TDP-43 mutant impairs endogenous TDP-43 function by sequestrating the endogenous TDP-43 into aggregates.(A to C) Cell viability and cytotoxicity of iPSC-derived motor neurons infected with lentivirus expressing TDP-43WT or TDP-436M. Experimental protocol (A), MTS assay (B), and LDH assay (C) (relative to mock-transfected cells). (D) Relative viability of Neuro2a cells transiently transfected with TDP-43, control siRNAs, or siRNA targeting TDP-43 (relative to mock-transfected cells) was determined by MTS assay. Triplicate samples were analyzed in three biologically independent experiments. (E) Exon skipping assay for CFTR exon 9. Neuro2a cells were transiently cotransfected with TDP-43 and CFTR minigene reporter plasmids. Splicing of CFTR exon 9 was assessed by RT-PCR (top). Quantification of the exclusion/inclusion ratio of CFTR exon 9 (relative to mock-transfected cells) was performed from the band intensities (bottom). n = 3, biologically independent experiments. (F to H) Quantification of RNA levels of Tardbp intron 7 (F), Sort1 (G), and Poldip3 (H) with indicated exon inclusion. Neuro2a cells were transiently transfected with TDP-43, control siRNAs, or siRNAs targeting TDP-43, and mRNA levels (relative to mock-transfected cells) were analyzed by quantitative RT-PCR. n = 4 biologically independent experiments. (I) Representative immunoblots showing endogenous TDP-43 and mCherry levels in cytoplasmic (Cyto), nuclear (Nuc), and 1% Triton X-100 insoluble fractions (Insol) of Neuro2a cells transiently expressing mCherry or TDP-436M-mCherry. GAPDH and fibrillarin were used as the cytoplasmic and nuclear markers, respectively. (J) Quantification of the percentage of endogenous TDP-43 in each fraction to the total endogenous TDP-43 in (I). n = 3, biologically independent experiments. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 [ANOVA with Tukey’s test in (B) to (H) and unpaired two-sided t test in (J)].

Fig 5: A bimolecular fluorescence complementation assay, TDP-DiLuc, demonstrates that transcription inhibition impairs TDP-43 dimerization/multimerization through spliceosomal defects.(A) Schematic illustration of TDP-DiLuc, a NanoBiT-based complementation assay to monitor the TDP-43 dimerization/multimerization. aa, amino acids. (B) Relative luminescence in the Neuro2a cells transiently expressing TDP-DiLuc WT or NDD mutants (normalized to mean TDP-43WT levels). Triplicate samples were analyzed in three biologically independent experiments. (C) Quantification of the TDP-43 dimerization under stress conditions. Relative DiLuc luminescence of Neuro2a cells stably expressing TDP-DiLuc WT treated with actinomycin D (ActD), MG-132, H2O2, sodium arsenite, sorbitol, or heat shock (relative to nontreated control cells). Triplicate samples were analyzed in three biologically independent experiments. (D) Representative confocal images of HeLa cells treated with the indicated reagents followed by staining with anti-SMN. Arrowheads indicate SMN-positive Gems. (E) Quantification of Gems per cell in (D). The number of Gems was quantified in 100 cells from three independent experiments. (F) Representative confocal images of SH-SY5Y cells transiently transfected with control siRNA or siRNA targeting SNRNP70. Cells treated with ActD (5 μg/ml; 3 hours) served as the positive control. Arrowheads indicate SMN-positive Gems. DMSO, dimethyl sulfoxide. (G) Quantification of Gems per cell in (F). The number of Gems was quantified in 100 cells from three biologically independent experiments. (H) Quantification of the relative DiLuc luminescence in SH-SY5Y cells transiently transfected with TDP-DiLuc and control siRNA or siRNA targeting SNRNP70. Cells treated with ActD (5 μg/ml; 3 hours) served as the positive control. Triplicate samples were analyzed in three biologically independent experiments. Scale bars, 20 μm (D and F). Data are expressed as means ± SEM (B, C, E, G, and H). ***P < 0.001 and ****P < 0.0001 [ANOVA with Tukey’s test in (B), (C), (E), (G), and (H)].