Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation

Abstract

Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA-binding protein TAR DNA-binding protein (TDP-43), is hyperphosphorylated in disease on several C-terminal serine residues, a process generally believed to promote TDP-43 aggregation. Here, we however find that Casein kinase 1δ-mediated TDP-43 hyperphosphorylation or C-terminal phosphomimetic mutations reduce TDP-43 phase separation and aggregation, and instead render TDP-43 condensates more liquid-like and dynamic. Multi-scale molecular dynamics simulations reveal reduced homotypic interactions of TDP-43 low-complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We speculate that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.

Keywords: RNA-binding protein; TDP-43; neurodegeneration; phase separation; phosphorylation.

Figure EV1. Identification of TDP‐43‐MBP‐His₆ phospho‐sites after in vitro phosphorylation with CK1δ

1. Identification of TDP‐43 phospho‐sites on in vitro phosphorylated TDP‐43 (+CK1δ, +ATP) in comparison to controls (−CK1δ −ATP; CK1δ only; ATP only) by Western blot. Samples were analyzed by SDS–PAGE and Western blot using a rabbit anti‐TDP‐43 N‐term antibody (Proteintech) to detect total TDP‐43, rat anti‐TDP‐43‐phospho Ser409/410 (clone 1D3, Helmholtz Center Munich) and mouse anti‐TDP‐43‐phospho Ser403/404 (Proteintech, Cat. No.: 66079‐1‐Ig) antibodies.

2. Schematic diagrams showing sequence coverage in mass spectrometry after trypsin digest (underlined) and phosphorylated serine/threonine residues (orange) of in vitro phosphorylated TDP‐43‐MBP‐His₆ with CK1δ + ATP (one out of two representative experiments is shown).

Source data are available online for this figure.

Figure 1. TDP‐43 phosphorylation by CK1δ and C‐terminal phosphomimetic substitutions reduce TDP‐43 condensation in vitro

1. A

   Scheme of sedimentation assay (created in BioRender.com): phase separation of TDP‐43 was induced by TEV protease cleavage of TDP‐43‐MBP‐His₆, and condensates were pelleted by centrifugation.

2. B

   Sedimentation assay to quantify condensation of unmodified TDP‐43 versus in vitro phosphorylated TDP‐43 (+CK1δ, +ATP) and controls (CK1δ or ATP only); TDP‐43 detected by Western blot (rabbit anti‐TDP‐43 N‐term). Due to incomplete TEV cleavage, some TDP‐43‐MBP‐His₆ remains present and co‐fractionates with cleaved TDP‐43, due to TDP‐43 self–self interaction.

3. C

   Quantification of band intensities of cleaved TDP‐43 shown as mean of Supernatant/(Supernatant + Condensate) (S/[S + C]) ratio of three independent experimental replicates (n = 3) ± SD. ***P < 0.0002 by one‐way ANOVA with Dunnett's multiple comparison test to Wt.

4. D

   Schematic diagram of TDP‐43 and sequence of C‐terminal region (aa. 370–414) for Wt, phosphomimetic (S‐to‐D) variants and control (S‐to‐A) variants. NTD, N‐terminal domain; RRM, RNA recognition motif; LCD, low complexity domain with α‐helical structure.

5. E

   Turbidity measurements (optical density [OD] at 600 nm) to quantify phase separation of the indicated TDP‐43 variants at three different concentrations (in Hepes buffer). Values represent mean of three independent experimental replicates (n = 3) ± SD. *P < 0.0332, **P < 0.0021 and ***P < 0.0002 by one‐way ANOVA with Dunnett's multiple comparison test to Wt, comparing the respective concentration condition (5, 10 and 20 µM).

6. F–I

   Representative bright field microscopic images of TDP‐43 condensates (in Hepes buffer), Bar, 25 µm (F) and quantification of condensate number (G), size (H) and roundness (I). Box plots show the comparison of median and inter‐quartile range (upper and lower quartiles) of all fields of view (FOV) from Min to Max (whiskers) of two replicates (≥ 22 FOV per condition). *P < 0.0332, **P < 0.0021 and ***P < 0.0002 by one‐way ANOVA with Dunnett's multiple comparison test to Wt, comparing the respective concentration condition (5, 10 and 20 µM).

Source data are available online for this figure.

Figure EV2. C‐terminal phosphomimetic substitutions reduce TDP‐43 condensation in vitro

1. Turbidity measurements (optical density [OD] at 600 nm) to quantify phase separation of different S‐to‐D and S‐to‐A mutants in comparison to TDP‐43 Wt using phosphate buffer. Values represent mean of three independent experimental replicates (n = 3) ± SD. *P < 0.0332, **P < 0.0021 and ***P < 0.0002 by one‐way ANOVA with Dunnett's multiple comparison test to Wt, comparing the respective concentration condition (5, 10, 20 µM).

2. Sedimentation assay to quantify condensation of different S‐to‐D mutants in comparison to TDP‐43 Wt (in Hepes buffer). TDP‐43 was detected by TDP‐43 Western blot (rabbit anti‐TDP‐43 N‐term).

3. Quantification of band intensities of cleaved TDP‐43 corresponding to supernatant (S) and condensates (C) fractions is shown as mean of S/(S + C) ratio of three independent experimental replicates (n = 3) ± SD. **P < 0.0021 and ***P < 0.0002 by one‐way ANOVA with Dunnett's multiple comparison test to Wt.

4. Representative bright field microscopic images of TDP‐43 condensates formed from TDP‐43 Wt vs different S‐to‐D or S‐to‐A variants in phosphate buffer (Bar, 25 µm).

Source data are available online for this figure.

Figure 2. C‐terminal phosphomimetic substitutions enhance liquidity of TDP‐43 condensates and reduce TDP‐43 aggregation in vitro

1. Representative still images of Alexa488‐labeled TDP‐43 condensates by spinning disc timelapse confocal microscopy. Wt condensates do not fuse, 5D condensates fuse slowly and 12D condensates readily fuse upon contact and relax into spherical droplets. Bar, 5 µm.

2. Representative images of FRAP experiments at indicated time‐points. Boxes indicate bleached area (half‐bleach of condensate). Bar, 5 µm.

3. FRAP curves after half‐bleach of freshly formed Alexa488‐labeled TDP‐43 condensates. Values represent mean ± SD of three independent experimental replicates (n = 3) of ≥ 9 droplets analyzed per condition. ***P < 0.0002 by one‐way ANOVA with Tukey's multiple comparison test for area under the curve (AUC) of individual droplets.

4. Confocal images of Alexa488‐labeled TDP‐43 aggregates formed in an in vitro aggregation assay (with TEV protease cleavage). Bar, 100 µm. Zoom shows magnified view of aggregates at the 24 h time point. Bar, 20 µm.

5. SDD‐AGE followed by TDP‐43 Western blot to visualize SDS‐resistant oligomers/high‐molecular‐weight species of TDP‐43‐MBP‐His₆ in an in vitro aggregation assay (without TEV protease cleavage). Asterisks represent monomeric (*), oligomeric (**) and polymeric (***) species.

6. Input of TDP‐43‐MBP‐His₆ variants used in the SDD‐AGE assay, detected by Western blot (anti‐TDP‐43 N‐term).

Source data are available online for this figure.

Figure EV3. Analysis of contacts in biomolecular condensates formed by the TDP‐43 LCD in coarse‐grained simulations

1. A, B

   Contact maps for Wt (A) and 12D (B) TDP‐43 LCD from simulations with the explicit solvent Martini coarse‐grained model. Residue i and residue j are defined to be in contact if any of the coarse‐grained beads are within 4.5 Å. The relative contact probability is calculated by averaging over all 118 protein chains and the last 5 of 20 μs simulations each. Intra‐chain contacts with the two preceding and following residues are excluded from the analysis. Aromatic residues form prominent contacts and are highlighted by black arrows. For example, looking at the column for F276 and following it upwards one can see that F276 interacts with F276 in other chains and irrespective of the chain, with F283, F289, F313, F316, W334, F367, Y347, W385, F401, and W412. The sites of the phosphomicking S‐to‐D mutations are highlighted by purple arrows. At these sites differences between Wt and 12D LCD can be seen, with Wt forming more contacts close in protein sequence and 12D instead interacting with R268, R272, R275, R293, and R361 further away in the sequence.

2. C

   Differences in contact probability P_i,j  = P_i,j (Wt) − P_i,j (12D) from simulations with the explicit‐solvent Martini coarse‐grained model. Differences highlight that wild‐type S residues, unlike phosphomicking D residues, favor interactions with residues close in sequence, while demonstrating that most contacts are not affected by the phosphomicking S‐to‐D mutations. Black and purple arrows correspond to aromatic residues and phosphomicking S‐to‐D mutations, respectively.

Figure 3. Atomistic and coarse‐grained simulations of TDP‐43 LCD: phosphomimicking residues form fewer protein–protein interactions and more protein–solvent interactions

1. A

   TDP‐43 LCD phase separates in coarse‐grained simulations with explicit solvent. Condensate of TDP‐43 Wt LCD is shown, protein colored according to chain identity. Water omitted for clarity. Ions shown in cyan.

2. B

   Normalized probability of protein‐protein contacts by phosphomimicking aspartates in 12D and serines in Wt resolved by amino acid type from coarse‐grained simulations. Error bars smaller than symbols. Inset: Distributions of the number of water molecules within 5 Å of side chains of phosphomimicking aspartates of 12D and corresponding serines in Wt from 15 µs of coarse‐grained molecular dynamics simulations.

3. C

   Atomistic simulation setup of 32 TDP‐43 LCDs. Different LCD chains shown in different colors in space‐filling representation. For one chain (lower left), a transparent surface reveals its atomic structure as sticks.

4. D

   Normalized probability of protein–protein contacts by phosphomimicking aspartates in 12D and serines in Wt resolved by amino acid type from atomistic simulations. Two 1 µs simulations are distinguished by color intensity. Inset: distributions of the number of water molecules within 5 Å of the side chains of phosphomimicking aspartates of 12D and the corresponding serines in Wt from atomistic simulations.

5. E

   Representative snapshots of atomistic simulations showing water within 3 Å of (left) Wt S407, S409 and S410 with nearby LCDs in surface representation and (right) 12D D407, D409 and D410. Protein surfaces are colored according to chain identity.

6. F

   Density profiles in TDP‐43 LCD condensates (peak at center) coexisting with dilute solutions for Wt, 12D, 5pS, 12pS and 12A from coarse‐grained simulations with the implicit solvent coarse‐grained HPS model.

7. G, H

   Snapshots of 12D condensate (G) and fragmented 12pS clusters (H) in simulations with the coarse‐grained HPS model. Side view on elongated boxes (blue lines).

Figure EV4. Phosphomimetic substitutions do not alter nuclear localization, UG‐rich RNA binding and autoregulation of TDP‐43

1. Immunostainings showing nuclear localization of myc‐TDP‐43 Wt, 12D and 12A in HeLa cells. Endogenous TDP‐43 expression was silenced by siRNAs, followed by transient transfection of the indicated siRNA‐resistant myc‐TDP‐43 constructs. After 24 h, localization of TDP‐43 Wt, 12D and 12A variants was visualized by TDP‐43 immunostaining (mouse anti‐TDP‐43 antibody, Proteintech). G3BP1 (rabbit anti‐G3BP1 antibody, Proteintech) and DAPI signal is shown to visualize the cytoplasm and nuclei, respectively. In the merge (right column), DAPI is show in turquoise, TDP‐43 in green, and G3BP1 in magenta. Bar, 30 µm.

2. Electrophoretic mobility shift assay (EMSA) of TDP‐43‐MBP‐His₆ variants (Wt, 12D and 12A) in a complex with (UG)₁₂ RNA.

3. Representative confocal images of U2OS cells stably expressing the indicated myc‐TDP‐43 variants (Wt, 12D and 12A) after siRNA KD of endogenous TDP‐43 and induction of myc‐TDP‐43 expression with doxycycline. Cells were stained with mouse monoclonal anti‐myc 9E10 antibody (IMB protein production facility) and DAPI. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), DAPI is shown in turquoise) and myc‐TDP‐43 is shown in green. Bar, 20 µm.

4. Western Blot showing the expression levels of myc‐TDP‐43 variants in stable inducible Flp‐In T‐Rex U2OS cell lines before and after addition of doxycycline (dox). Samples were analyzed by SDS–PAGE and Western blot using a rabbit anti‐TDP‐43 N‐term antibody (Proteintech, upper blot), mouse anti‐myc 9E10 antibody (IMB protein production core facility), and rabbit anti‐Histone H3 antibody (Abcam) to detect the loading control Histone H3.

5. Quantification of TDP‐43 autoregulation after dox‐induced expression of myc‐TDP‐43 variants in U2OS cell lines. Values represent the mean ± SD of four independent experimental replicates (n = 4) of endogenous TDP‐43 expression levels normalized to Wt (−Dox) condition. *P < 0.0332 and ***P < 0.0002 by one‐way ANOVA with Šídák's multiple comparisons test of TDP‐43 endogenous expression levels, comparing the respective non‐induced (−Dox) and induced (+Dox) lines.

Source data are available online for this figure.

Figure 4. Phosphomimetic substitutions do not alter the rate of TDP‐43 nuclear import and do not impair TDP‐43 autoregulation, RNA‐binding or alternative splicing function

1. Hormone‐inducible nuclear import assay, representative still images of GCR₂‐EGFP₂‐TDP‐43 Wt, 12D and 12A before and during import triggered by addition of dexamethasone. Images were live recorded by spinning disc confocal microscopy. Bar, 20 µm.

2. Quantification of the hormone‐inducible nuclear import measured during a total time course of 50 min. Values represent the mean fluorescence intensity of GCR₂‐EGFP₂‐TDP‐43 in the cytoplasm for three independent replicates ± SEM (≥ 42 cells per condition).

3. Phosphomimetic 12D TDP‐43 is competent in autoregulating TDP‐43 expression. SDS–PAGE followed by TDP‐43 Western blot showing downregulation of endogenous TDP‐43 through autoregulation (60) after 48 h expression of Wt, 12D and 12A variants in HeLa cells. TDP‐43 was detected using rabbit anti‐TDP‐43 C‐term antibody (Proteintech), Histone H3 (rabbit anti‐Histone H3 antibody, Abcam) was visualized as a loading control. * denotes an unspecific band.

4. Electrophoretic mobility shift assays (EMSA) of TDP‐43‐MBP‐His₆ variants (Wt, 12D and 12A) in a complex with TDP‐43 autoregulatory RNA binding site (60). All TDP‐43 variants form TDP‐43‐RNA complexes equally well.

5. Splicing analysis by RT–PCR of known TDP‐43 splice targets (SKAR exon 3 and Bim exon 3) in HeLa cells. Silencing of endogenous TDP‐43 by siRNA leads to altered splice isoforms of SKAR and Bim (second vs first lane). These splicing alterations can be rescued by re‐expression of TDP‐43 Wt, but also 12D or 12A variants, demonstrating that phosphomimetic TDP‐43 is fully competent in regulation splicing of these TDP‐43 splice targets.

Source data are available online for this figure.

Figure 5. Phosphorylation and phosphomimetic substitutions reduce recruitment of TDP‐43 into stress‐induced membrane‐less organelles

1. Scheme of stress granule (SG) recruitment assay in semi‐permeabilized cells.

2. Reduced SG association of TDP‐43 by 12D mutations or in vitro phosphorylation. Bar, 20 µm.

3. Quantification of TDP‐43‐MBP‐His₆ mean fluorescence intensity in SGs normalized to Wt ± SEM of three independent experimental replicates (n = 3; ≥ 10 cells; ≥ 46 SGs each). **P < 0.0021 and ***P < 0.0002 by one‐way ANOVA with Dunnett's multiple comparison test to Wt.

4. SG recruitment of TDP‐43 variants in intact HeLa cells in absence of endogenous TDP‐43. After TDP‐43 silencing and expression of myc‐TDP‐43 Wt, 12D and 12A variants, SGs were induced by H₂O₂ treatment and SG recruitment of TDP‐43 was monitored by TDP‐43 and TIA1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei were stained in DAPI (turquoise), TDP‐43 (green) and TIA‐1 (magenta). Bar, 25 µm.

5. Quantification of TDP‐43 in SGs versus cytoplasm ± SD of two independent experimental replicates (n = 2; ≥ 62 cells; ≥ 234 SGs each). ***P < 0.0002 by Krustal–Wallis test with Dunn's multiple comparison test to Wt.

6. SG recruitment of different TDP‐43‐NLSmut variants in intact HeLa cells in the absence of endogenous TDP‐43. After TDP‐43 silencing and expression of NLSmut Wt, 12D and 12A variants, SGs were induced by H₂O₂ treatment and SG recruitment of TDP‐43 was monitored by TDP‐43 and G3BP1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei were stained in DAPI (turquoise), TDP‐43 (green) and G3BP1 (red). Bar, 40 µm.

7. Quantification of TDP‐43‐NLS mutants in SGs versus band around SGs of two independent replicates ± SD. ***P < 0.0002 by Kruskal–Wallis test with Dunn's multiple comparison test to Wt (≥ 56 cells; ≥ 315 SGs per condition).

8. Recruitment of TDP‐43 into arsenite‐induced nuclear bodies (NBs) in HeLa cells. After TDP‐43 silencing and expression myc‐TDP‐43 Wt, 12D and 12A, NBs were induced by sodium arsenite treatment and NB recruitment of TDP‐43 was monitored by TDP‐43 immunostaining. Bar, 20 µm.

9. Percentage of cells with TDP‐43 in NBs ± SD of three independent experimental replicates (n = 3). *P < 0.0332 by Kruskal–Wallis test with Dunn's multiple comparison test to Wt.

10. Intensity profiles (right) of TDP‐43 Wt, 12D and 12A variants (green) along white lines (left). Bar, 10 µm.

Figure EV5. Phosphomimetic S‐to‐D substitutions reduce association of TDP‐43 with stress granules and nuclear stress bodies

1. Association of TDP‐43 with stress granules (SGs) in semi‐permeabilized HeLa cells is suppressed by phosphomimetic (2D, 5D and 12D) mutations in comparison to TDP‐43 Wt and 12A. SGs and TDP‐43‐MBP‐His₆ were visualized by G3BP1 and MBP immunostaining, respectively. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), G3BP1 is shown in magenta, TDP‐43‐MBP‐His₆ in green, white pixels indicate colocalization. Nuclei were counterstained with DAPI (turquoise). Bar, 10 µm.

2. Quantification of the mean fluorescence intensity of TDP‐43‐MBP‐His₆ in SGs normalized to Wt for four independent replicates ± SEM, ***P < 0.0002 defined by 1‐way ANOVA with Dunnett's multiple comparison test (≥ 10 cells; ≥ 46SGs per condition). Bkgr = background fluorescence in the green channel.

3. Representative confocal images of U2OS cells stably expressing the indicated myc‐TDP‐43 variants (Wt, 12D and 12A) after siRNA KD of endogenous TDP‐43 and induction of myc‐TDP‐43 expression with doxycycline, followed by sodium arsenite stress (2 h) to induce nuclear stress bodies (NBs). Cells were stained with mouse monoclonal anti‐myc 9E10 antibody (IMB protein production facility) and DAPI. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), DAPI is shown in turquoise and myc‐TDP‐is shown in green. Bar, 20 µm.

4. Intensity profiles (right) of myc‐TDP‐43 Wt, 12D and 12A variants (green), expressed in Flp‐In T‐Rex USOS stable cell lines, along white lines (left). Bar, 10 µm.

Figure 6. Phosphomimetic substitutions enhance TDP‐43 solubility in HeLa cells and primary neurons

1. Biochemical fractionation into RIPA‐soluble (S) and RIPA‐insoluble (I) fractions to analyze solubility of the different myc‐TDP‐43 variants (Wt, 12D and 12A) expressed in HeLa cells for 48 h. TDP‐43 was detected by TDP‐43 Western blot (upper blot, rabbit anti‐TDP‐43 C‐term, Proteintech) and Myc Western blot (lower blot, mouse anti‐Myc 9E10).

2. Quantification of myc‐TDP‐43 variants (Wt, 12D and 12A) in (S) versus (I) fractions extracted from TDP‐43 Western blots of four independent replicates ± SD, plotted as S/(S + I). *P < 0.0332 by one way ANOVA with Dunnett's multiple comparison test to Wt.

3. RIPA‐insoluble material of the indicated EGFP‐tagged TDP‐43 variants (± NLS mutation) expressed in primary cortical neurons analyzed by filter‐trap assay.

4. Primary hippocampal neurons expressing EGFP‐TDP‐43 Wt, 12D or 12A with additional NLS mutation. Bar, 80 µm. Right: zoomed images of white squares (TDP‐43 signal). Bar, 10 µm.

5. SG recruitment of EGFP‐TDP‐43 NLS mutant variants (Wt, 12D, 12A) in primary hippocampal neurons. SG formation was induced by 1 h heat shock at 42°C. SGs and TDP‐43 were monitored by G3BP1 antibody staining and EGFP fluorescence, respectively. For clarity, signals were converted to grey values in the individual channels (first two columns). In the merge (third column), EGFP‐TDP‐43 shown in green, G3BP1 in red and nuclei (DAPI staining) in turquoise. Bar, 20 µm.

Source data are available online for this figure.