Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation - PubMed (original) (raw)

doi: 10.1038/s41467-017-00062-0.

Eva-Maria Hock 1, Patrick Ernst 2, Chiara Foglieni 3, Melanie Jambeau 1, Larissa A B Gilhespy 1, Florent Laferriere 1, Zuzanna Maniecka 1, Andreas Plückthun 2, Peer Mittl 2, Paolo Paganetti 3, Frédéric H T Allain 4, Magdalini Polymenidou 5

Affiliations

Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation

Tariq Afroz et al. Nat Commun. 2017.

Abstract

TDP-43 is a primarily nuclear RNA-binding protein, whose abnormal phosphorylation and cytoplasmic aggregation characterizes affected neurons in patients with amyotrophic lateral sclerosis and frontotemporal dementia. Here, we report that physiological nuclear TDP-43 in mouse and human brain forms homo-oligomers that are resistant to cellular stress. Physiological TDP-43 oligomerization is mediated by its N-terminal domain, which can adopt dynamic, solenoid-like structures, as revealed by a 2.1 Å crystal structure in combination with nuclear magnetic resonance spectroscopy and electron microscopy. These head-to-tail TDP-43 oligomers are unique among known RNA-binding proteins and represent the functional form of the protein in vivo, since their destabilization results in loss of alternative splicing regulation of known neuronal RNA targets. Our findings indicate that N-terminal domain-driven oligomerization spatially separates the adjoining highly aggregation-prone, C-terminal low-complexity domains of consecutive TDP-43 monomers, thereby preventing low-complexity domain inter-molecular interactions and antagonizing the formation of pathologic aggregates.TDP-43 aggregation is observed in amyotrophic lateral sclerosis. Here the authors combine X-ray crystallography, nuclear magnetic resonance and electron microscopy studies and show that physiological oligomerization of TDP-43 is mediated through its N-terminal domain, which forms functional and dynamic oligomers antagonizing pathologic aggregation.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1

Fig. 1

TDP-43 oligomers are expressed in human cells and brain and resist oxidative stress. a Immunoblots (from 12% denaturing polyacrylamide gels) of human fibroblast fractions obtained upon incubation with increasing concentration of DSG cross-linker followed by nucleo-cytoplasmic fractionation. In the upper panel, high molecular weight bands (marked with green asterisks) are detected with anti-human TDP-43 antibody at increasing concentrations of DSG in the nuclear fraction (N) and in total cell lysates (T), but not in the cytoplasmic fraction (C). Lower panels show immunoblots for cytoplasmic marker GAPDH, or nuclear marker histone H3. Immunoblots are representative of three independent experiments. b Immunoblots (from 12% denaturing polyacrylamide gels) of human cerebellum samples. High molecular weight TDP-43 bands are detected in 1 mM DSG cross-linked samples (green asterisks) compared to samples obtained in the absence of cross-linker. Immunoblots are representative of three independent experiments. Full immunoblots with molecular weight marker are shown in Supplementary Fig. 1. c Schematic diagram depicting the experimental set-up for oxidative stress treatment in mouse organotypic slice cultures followed by DSG mediated cross-linking. Subset of images (such as eppendorf tubes and microscope) in the schematic figure were adapted from the Servier medical art (

http://www.servier.com/slidekit

). d Confocal microscopy images of non-treated (control) and arsenite-treated (2 h Arsenite) mouse brain slices, immunostained with antibodies against TDP-43 and TIA-1. Cytoplasmic TDP-43 granules (red) co-localize with the stress-granule marker TIA-1 (green). Merged images including DAPI-staining of nuclei (blue) are shown in the right panels. e Immunoblots (from 12% denaturing polyacrylamide gels) of control (NT) and arsenite-treated (As) mouse brain slices after cross-linking by DSG and nucleo-cytoplasmic fractionation. DSG-cross-linked oligomeric TDP-43 (marked with green asterisks) remains localized in the nuclear fraction after oxidative stress. Immunoblots are representative of three independent experiments

Fig. 2

Fig. 2

Crystal structure of TDP-43 NTD at 2.1 Å resolution. a Schematic domain organization of human TDP-43 with the N-terminal domain (NTD) shown in blue, RNA recognition motifs (RRMs) in gray and the low-complexity domain (LCD) in red with domain boundaries indicated on top. b Crystals of TDP-43 NTD show helical filaments with single molecules arranged in head-to-tail fashion as seen from the side. Atoms of Mol-A and -B (comprising asymmetric unit) are shown as cyan and blue spheres, respectively. The dimensions of the crystallographic helix are indicated. c Wall-eyed stereo view of two TDP-43 NTD molecules (Mol-A, Mol-B) in the asymmetric unit. The two molecules are shown in cartoon representation with the secondary structure elements and the N- and C-termini of the molecules labeled. The side chains making inter-molecular contacts are shown in stick representation in the corresponding domain color and labeled. Inter-molecular hydrogen bonds are shown as dotted magenta lines. Similar color and labeling schemes are used in other figures unless stated. d, e Electrostatic charge on the surface of two TDP-43 NTD molecules (Mol-A, Mol-B) of the asymmetric unit. Amino-acid residues in the positively charged head d and negatively charged tail region e are shown in stick representation and labeled. Positive potential is shown in blue and negative potential in red

Fig. 3

Fig. 3

TDP-43 NTD oligomerization analyzed by solution NMR spectroscopy. a Overlay of 2D 1H-15N HSQC spectra at 30 °C of 15N isotopically labeled TDP-43 NTD at protein concentrations of 50, 100, 200, 400, and 800 μM in red, green, blue, magenta, and black, respectively. Magnified views of cross peaks undergoing chemical shift perturbations (CSP) are shown around the full spectra overlay while peaks showing line broadening are encircled in green and labeled. Black arrows show the direction of movement of the peaks. b Backbones of amino acids undergoing CSP are colored on the cartoon of TDP-43 NTD crystal structure. Relative to the lowest protein concentration (50 μM), the changes in average chemical shift perturbations (ΔCSP = Δ((δH)2 + (δN/5)2)/2)1/2, in which δH and δN are in ppm) were calculated at highest protein concentration (800 μM) and colored on the structure based on the magnitude of perturbations as indicated. Side chains contributing to inter-molecular interface seen in the crystal structure are displayed as sticks with H-bonds in magenta

Fig. 4

Fig. 4

Point mutations at oligomerization interface abrogate TDP-43 polymerization in vitro and in cells. a Transmission electron microscopy (TEM) image of TDP-43 NTD fibrillar oligomers following negative staining. One region (dotted black box) from the image is shown magnified on the right. b Position of the six amino acids that were substituted to abolish oligomerization are shown on the cartoon of TDP-43 NTD crystal structure with side chains shown as sticks and labeled in red. c Overlay of 2D 1H-15N HSQC spectra of wild type TDP-43 NTD (blue) and oligomerization mutant TDP-43 NTD-6M (red) at protein concentration of 100 μM at 30 °C. d Overlay of 2D1H-15N HSQC spectra of TDP-43 oligomerization mutant TDP-43 NTD-6M at increasing protein concentration of 100, 200, 400, and 800 μM in red, green, magenta and black, respectively at 30 °C. e TEM image showing the lack of fibrillar structures for the oligomerization mutant TDP-43 NTD-6M. One region (dotted black box) from the image is shown magnified on the right. f Immunoblots (from 4–12% denaturing polyacrylamide gel) with anti-GFP antibody (upper panel) of lysates obtained from cells transiently expressing wild-type GFP-tagged human TDP-43 (GFP-TDP-43) or oligomerization mutants (GFP-TDP-43-Tm/Hm/4 M/5 M/6 M/ΔNTD) followed by DSG-mediated cross-linking. Oligomerization mutants display significantly reduced high-molecular weight oligomeric TDP-43 compared to wild-type TDP-43. Actin is used as protein loading control (lower panel). Immunoblots are representative of three independent experiments

Fig. 5

Fig. 5

TDP-43 oligomerization is essential for its role in RNA metabolism. a Schematic overview of rescue experiment to assess the role of TDP-43 oligomerization in RNA splicing. Point mutations interfering with TDP-43 oligomerization are schematically marked by red asterisks below the NTD. b Immunoblot (from 12% denaturing polyacrylamide gel) using an antibody against mouse/human TDP-43, showing specific downregulation of endogenous mouse TDP-43 and overexpression of RNAi resistant GFP-tagged wild type and oligomerization mutant human TDP-43 (GFP-TDP-43-4M/5 M/6 M) in mouse NSC-34 cells (upper panel). Actin is used as protein loading control (lower panel). Immunoblots are representative of four independent experiments. c Semi-quantitative RT-PCR analysis of selected target’s alternative splicing regulated by TDP-43. Left panel depicts alternatively spliced exons (orange) or cryptic exons (red) flanked by their constitutive exons in blue boxes. Middle panel shows representative polyacrylamide gel images of semi-quantitative RT-PCR products for all conditions. Right panel shows quantification of splicing changes plotted as the ratio of exon inclusion/exclusion (on _y_-axis) against different conditions (on _x_-axis) averaged from four biological replicates and normalized to the ratio obtained for not treated (NT) cells that is arbitrarily set to 1. In all cases, the expression of wild-type TDP-43 (green bars) rescues the changes in splicing caused by downregulation of endogenous mouse TDP-43 (TDP-43 si-RNA, black bars). The effect of each oligomerization mutant TDP-43 is shown (red bars). Statistical comparison of the inclusion/exclusion ratio between wild type and oligomerization mutants (4 M, 5 M, or 6 M) is indicated by asterisks above respective bars (*p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student _t_-test). Standard deviation is represented by error bars. Full gels with molecular weight markers are shown in Supplementary Fig. 10

Fig. 6

Fig. 6

NTD-mediated TDP-43 oligomerization impedes inter-molecular LCD interactions and antagonizes pathologic protein aggregation. a Schematic of tripartite GFP complementation assay. TDP-43 was tagged either at the N- or C-terminus with the 10th (T10) or 11th (T11) β-strand of GFP (schematically indicated in magenta and light yellow, respectively). Successful complementation results in fluorescence reconstitution with GFP1-9 molecule (GFP β-strands 1–9 shown in blue and α-helix in grey). b, c N- b or C-terminally c tagged T10- and T11- TDP-43 were co-transfected with GFP1–9 in mouse C17.2 cells. Confocal microscopy analysis on cells shows successful complementation as fluorescence reconstitution in case of N-terminally tagged T10- and T11- TDP-43 b. In contrast, the C-terminal tagged T10- and T11- TDP-43 fail to reconstitute fluorescence c. Merged and zoomed in images are shown on the right. Transfected mouse C17.2 cells were counter-stained with a human-specific TDP-43 antibody (red) and the nuclear marker DAPI (blue). d–f Confocal microscope images of NSC-34 cells overexpressing GFP-tagged wild type d or oligomerization defective TDP-43 mutants e, f. Cells were stained with anti-GFP (green), anti-TDP-43 (red) and anti-phosphorylated TDP-43 (gray) antibodies. Merged images overlaid with DAPI (blue) are shown on the right. g The percentage of phosphorylated TDP-43 (pTDP-43)-positive cells is plotted for wild-type and oligomerization mutant TDP-43 (TDP-43-6M and TDP-43-ΔNTD). Manual cell counting was conducted on confocal images (20× magnification). For each condition, approximately 2500 GFP-positive cells (or 1500 cells for GFP-TDP-43 ΔNTD) from three biological replicates were counted by an investigator that was blinded to the identity of the samples. Percentage of phosphorylation was determined considering the total number of GFP-positive transfected cells. An unpaired student _t_-test was performed to determine significance (depicted as asterisks). _P_-values: WT vs. 6 M: <0.0001 (****), WT vs. ΔNTD: 0.003 (**), 6 M vs. ΔNTD: 0.184 (n.s)

Fig. 7

Fig. 7

Novel TDP-43 oligomerization domain and implications for disease. a Multiple sequence alignment of TDP-43 NTD with structurally homologous DIX domains (Axin-1, Dvl-2) and Ubiquitin. Alignment was performed by T-coffee (

http://www.ebi.ac.uk/Tools/msa/tcoffee/

) and the alignment figure was generated in Jalview (

http://www.jalview.org/

). TDP-43 NTD secondary structural elements are depicted above the sequence and residues involved in the inter-molecular interactions within TDP-43 NTD are marked by red asterisks below the sequence. Sequence conservation scores are shown below the alignment as color gradient (_dark yellow_—poor conservation, _light yellow_—high conservation). b Overlay of structures of TDP-43 NTD (in blue) with Axin-1 DIX (in magenta, PDB ID 1WSP), Dvl2-DIX (in green, PDB ID 4WIP), and Ubiquitin (in yellow, PDB ID 1TBE). Structures are overlaid on one molecule to reveal the differences in the orientation of the second molecule. Individual structures in the same orientation are shown around the overlay. c Role of dynamic nuclear TDP-43 polymerization in physiological conditions and in disease. Within the nucleus, TDP-43 oligomerizes via the NTD and such conformation prevents irreversible aggregation by spatially separating the LCDs of adjoining TDP-43 molecules. Disturbance in the equilibrium between the oligomeric and monomeric TDP-43 in the cytoplasm (shown by green-red arrows) may result in proteolytic cleavage of exposed monomeric TDP-43 and generation of C-terminal fragments lacking the NTD. These highly aggregative fragments may initiate formation of irreversible pathologic inclusions via the LCD

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