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Structure of pathological TDP-43 filaments from ALS with FTLD

Nature volume 601, pages 139–143 (2022)Cite this article

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Abstract

The abnormal aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in neurons and glia is the defining pathological hallmark of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and multiple forms of frontotemporal lobar degeneration (FTLD)1,2. It is also common in other diseases, including Alzheimer’s and Parkinson’s. No disease-modifying therapies exist for these conditions and early diagnosis is not possible. The structures of pathological TDP-43 aggregates are unknown. Here we used cryo-electron microscopy to determine the structures of aggregated TDP-43 in the frontal and motor cortices of an individual who had ALS with FTLD and from the frontal cortex of a second individual with the same diagnosis. An identical amyloid-like filament structure comprising a single protofilament was found in both brain regions and individuals. The ordered filament core spans residues 282–360 in the TDP-43 low-complexity domain and adopts a previously undescribed double-spiral-shaped fold, which shows no similarity to those of TDP-43 filaments formed in vitro3,4. An abundance of glycine and neutral polar residues facilitates numerous turns and restricts β-strand length, which results in an absence of β-sheet stacking that is associated with cross-β amyloid structure. An uneven distribution of residues gives rise to structurally and chemically distinct surfaces that face external densities and suggest possible ligand-binding sites. This work enhances our understanding of the molecular pathogenesis of ALS and FTLD and informs the development of diagnostic and therapeutic agents that target aggregated TDP-43.

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Fig. 1: TDP-43 pathology of ALS with FTLD.
Fig. 2: Cryo-EM maps and atomic model of TDP-43 filaments from ALS with FTLD.
Fig. 3: Surfaces of TDP-43 filaments from ALS with FTLD.

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Data availability

Cryo-EM datasets have been deposited to the Electron Microscopy Public Image Archive (EMPIAR) under the following accession numbers: 10830 for individual one, frontal cortex; 10831 for individual one, motor cortex; and 10832 for individual two, frontal cortex. Cryo-EM maps have been deposited to the Electron Microscopy Data Bank (EMDB) under the following accession numbers: 13708 for individual one, frontal cortex; 13710 for individual one, motor cortex; and 13712 for individual two, frontal cortex. The atomic model of the double-spiral fold has been deposited to the Protein Data Bank (PDB) under accession number 7PY2. The atomic model of the α-subunit of glutamate synthase was obtained from the PDB (accession number 1EA0). The atomic model of the α-helices formed in solution by the hydrophobic region of TDP-43 was obtained from the PDB (accession number 2N3X). The atomic models of TDP-43 filaments formed in vitro were obtained from the PDB (accession numbers 7KWZ, 6N3C, 6N3A, 6N37 and 6N3B). Mass spectrometry data have been deposited to the Japan Proteome Standard Repository (jPOSTrepo) under accession number PXD029001. Any other data are available from the corresponding author upon request.

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Acknowledgements

We thank the individuals and their families for donating the brain tissue; K. Yamada and Y. Itoh for the clinical information; A. Akagi for dissecting the brain tissue; T. Arai and H. Akiyama for helpful comments on the clinical history and neuropathology; R. Otani and M. Takase for technical support with immunohistochemistry; K. Yamashita and G. N. Murshudov for help with Servalcat; staff at the MRC Laboratory of Molecular Biology Electron Microscopy Facility for access to and support with EM; staff at the MRC Laboratory of Molecular Biology Scientific Computing Facility for access to and support with computing; and M. Goedert, S. H. W. Scheres, S.W. Davies, S. Tetter, T. S. Behr and R. R. Chen for discussions. D.A. is a Fellow at Darwin College, University of Cambridge. This work was supported by the Medical Research Council (MRC) grant MC_UP_1201/25 and Alzheimer’s Research UK award ARUK-RS2019-001 to B.R.-F.; the Japan Agency for Medical Research and Development (AMED) grants JP20ek0109392 and JP20ek0109391 to M.Y. and JP20dm0207072 to M.H; and the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) grant JPMJCR18H3 to M.H.

Author information

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Diana Arseni, Alexey G. Murzin & Benjamin Ryskeldi-Falcon

  2. Department of Brain and Neurosciences, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

    Masato Hasegawa & Fuyuki Kametani

  3. Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

    Makoto Arai

  4. Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan

    Mari Yoshida

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  1. Diana Arseni
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Contributions

M.Y. identified the individuals and performed the neuropathological examinations. M.A. performed the genetic analysis. M.H. extracted the TDP-43 filaments and performed immunohistochemistry, immunoblotting and immuno-EM. F.K. performed the mass spectrometry. D.A. performed cryo-EM. D.A. and B.R.-F. analysed the cryo-EM data. D.A., B.R.-F. and A.G.M. built and analysed the atomic model. B.R.-F. supervised the study. B.R.-F., M.Y. and M.H. are the senior authors. All authors contributed to writing the manuscript.

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Correspondence to Benjamin Ryskeldi-Falcon.

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The authors declare no competing interests.

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Peer review information Nature thanks James Shorter, Henning Stahlberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 TDP-43 pathology of ALS with FTLD continued.

a, Staining of TDP-43 (brown) in the frontal and motor cortices and spinal cords of individuals 1 and 2 with anti-TDP-43 (amino acids 203–209) antibody. Nuclei were counterstained in blue. Scale bars, 50 μm. b, Staining of TDP-43 glial cytoplasmic inclusions (brown) in the motor cortices of individuals 1 and 2 with anti-phosphorylated S409 and S410 TDP-43 antibody. Nuclei were counterstained in blue. Scale bars, 50 μm. c, d, Immunoblots of the total homogenate (T), Sarkosyl-soluble fraction (S) and Sarkosyl-insoluble fraction (I) from the motor cortices of individuals 1 and 2 with anti-TDP-43 (amino acids 203–209) (c) and anti-phosphorylated S409 and S410 TDP-43 (d) antibodies. The original, uncropped blots are shown in Supplementary Fig. 1. e, Immuno-electron microscopy of the Sarkosyl-insoluble fraction from the frontal cortex of individual 2 using anti-phosphorylated S409 and S410 TDP-43 antibody. Scale bar, 100 nm. a–e, Similar results were obtained in at least three independent experiments.

Extended Data Fig. 2 Cryo-EM and helical reconstruction.

a, Representative cryo-EM images of TDP-43 filaments from the frontal and motor cortices of individual 1 and from the frontal cortex of individual 2. Scale bars, 100 nm. Similar results were obtained in at least three independent experiments. b, Fourier shell correlation (FSC) curves for two independently-refined cryo-EM half-maps. The FSC threshold of 0.143 is shown with red dashed lines. c, Local resolution estimates for the Cryo-EM 3D reconstructions. Scale bars, 10 Å. d, Cryo-EM density maps viewed along the helical axis. Scale bars, 10 Å.

Extended Data Fig. 3 Cryo-EM density map and atomic model comparisons.

a–c, Views of the cryo-EM density map of TDP-43 filaments from the frontal cortex of individual 1 and the corresponding atomic model showing representative densities for amino acid side chains (a), peptide group oxygen atoms (b) and ordered solvent molecules (red arrows) (c). d, Fourier shell correlation (FSC) curves for the refined atomic model against the cryo-EM density map (black); for the atomic model shaken and refined using the first half-map against the first half-map (magenta); and for the same atomic model against the second half-map (green). The FSC threshold of 0.5 is shown with a red dashed line.

Extended Data Fig. 4 Double-spiral fold of TDP-43 filaments from ALS with FTLD.

a, Schematic view of the double-spiral fold. The two hydrophobic clusters in the hydrophobic nucleus, composed of the side chains of M307, M311, F313, M322, M323, A326, A329 and L330 (cluster 1); and A328, W334, M336 and L340 (cluster 2), are numbered. b, Hydrophobicity of the double-spiral fold from most hydrophilic (cyan) to most hydrophobic (yellow). c, Secondary structure of the double-spiral fold, depicted as five successive rungs. The glycine-rich (G282-G310, magenta), hydrophobic (M311-S342, white) and Q/N-rich (Q343-Q360, green) regions are highlighted. d, Views of the double-spiral fold, depicted as three successive rungs, showing hydrogen bonds (blue dashed lines) between buried polar side chains and main chain peptide groups. e, Superposition of residues N319-A326 of the double-spiral fold (cyan), depicted as two successive rungs, with residues G88-M95 and G107-A114 of the β-helix domain of glutamate synthase (magenta, PDB ID 1EA0). f, Unmasked cryo-EM 3D reconstructions of TDP-43 filaments from the frontal and motor cortices of individual 1 and from the frontal cortex of individual 2, shown as central slices perpendicular to the helical axis. Additional density within the prominent groove on the filament surface formed by the main chain of G282–Q286 and the side chain of Q286 is indicated with a magenta arrow. Additional densities adjacent to flat strips on the surface of the glycine-rich spiral branch between R293 and A315 are indicated with yellow arrows. Additional less well-resolved density projecting from a polar patch formed by the side chains of N352, Q354 and Q356 on the surface of the Q/N-rich spiral branch is indicated with a cyan arrow. Scale bars, 25 Å.

Extended Data Fig. 5 Comparison of the double-spiral fold with recombinant TDP-43 structures.

a, Amino acid sequence alignment of the secondary structure elements of the double-spiral fold and recombinant TDP-43 structures with the TDP-43 LC domain. PDB IDs are given for the recombinant TDP-43 structures. The glycine-rich (G282-G310, magenta), hydrophobic (M311-S342, white) and Q/N-rich (Q343-Q360, green) regions are highlighted on the double-spiral fold secondary structure elements. b, Secondary structure of the double-spiral fold and recombinant TDP-43. PDB IDs are given for the recombinant TDP-43 structures. The glycine-rich (G282-G310, magenta), hydrophobic (M311-S342, white) and Q/N-rich (Q343-Q360, green) regions are highlighted.

Extended Data Fig. 6 Heat stability of TDP-43 filaments from ALS with FTLD.

Representative cryo-EM images of TDP-43 filaments from the frontal cortex of individual 1 with and without heating at 65 °C for 10 min. Scale bars, 100 nm. Similar results were obtained in at least three independent experiments.

Extended Data Table 1 Clinicopathological evaluation and TDP-43 inclusion pathology
Full size table
Extended Data Table 2 Cryo-EM data collection, refinement and validation statistics
Full size table
Extended Data Table 3 Mass spectrometry analyses of post-translational modifications
Full size table
Extended Data Table 4 Compatibility of ALS-associated TARDBP mutations with the double-spiral fold
Full size table

Supplementary information

Supplementary Figure

Uncropped images of immunoblots presented in this study.

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Arseni, D., Hasegawa, M., Murzin, A.G. et al. Structure of pathological TDP-43 filaments from ALS with FTLD. Nature 601, 139–143 (2022). https://doi.org/10.1038/s41586-021-04199-3

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