Structural polymorphism of the low-complexity C-terminal domain of TDP-43 amyloid aggregates revealed by solid-state NMR

Abstract

Aberrant aggregation of the transactive response DNA-binding protein (TDP-43) is associated with several lethal neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Cytoplasmic neuronal inclusions of TDP-43 are enriched in various fragments of the low-complexity C-terminal domain and are associated with different neurotoxicity. Here we dissect the structural basis of TDP-43 polymorphism using magic-angle spinning solid-state NMR spectroscopy in combination with electron microscopy and Fourier-transform infrared spectroscopy. We demonstrate that various low-complexity C-terminal fragments, namely TDP-13 (TDP-43_300-414), TDP-11 (TDP-43_300-399), and TDP-10 (TDP-43_314-414), adopt distinct polymorphic structures in their amyloid fibrillar state. Our work demonstrates that the removal of less than 10% of the low-complexity sequence at N- and C-termini generates amyloid fibrils with comparable macroscopic features but different local structural arrangement. It highlights that the assembly mechanism of TDP-43, in addition to the aggregation of the hydrophobic region, is also driven by complex interactions involving low-complexity aggregation-prone segments that are a potential source of structural polymorphism.

Keywords: TDP-43; amyloid; amyotrophic lateral sclerosis; frontotemporal dementia; low-complexity domain; polymorphism; solid-state NMR.

FIGURE 1

Sequences of TDP-43 and various low-complexity C-terminal domain fragments used in this study. Boxes are used to denote different functional/structural domains. The nomenclature of the different CTD fragments is chosen to be consistent with (Mompean et al., 2016b; Shenoy et al., 2019).

FIGURE 2

Structural analysis of low complexity CTFs aggregates. (A–C) Negatively stained electron micrographs of TDP-13 (A), TDP-11 (B), and TDP-10 (C) aggregates. Scale bars (in yellow) are 200 nm. (D–F) X-ray diffraction patterns of TDP-13 (D), TDP-11 (E), and TDP-10 (F) aggregate display characteristic cross-β reflections. Reflections at 4.7 Å and 9–10 Å are highlighted.

FIGURE 3

Detection of protein segments associated with high mobility by MAS NMR. ¹H-¹³C INEPT experiments of TDP-13 (in brown) (A), TDP-11 (in orange) (B), and TDP-10 (in pink) (C) amyloid aggregates. The data were recorded at a ¹H frequency of 600 MHz, 11 kHz MAS at 278 K.

FIGURE 4

Comparison of the rigid cores of TDP-10, TDP11, and TDP-13 CTF aggregates by MAS NMR and FT-IR spectroscopy. (A–C) Spectral excerpts from the overlay of 2D¹³C–¹³C PDSD spectra (mixing time of 50 ms) of (A) TDP-13 (brown), (B) TDP-11 (orange), and (C) TDP-10 (pink) compared with TDP-16 (blue). Spectra were recorded at 600 MHz, 11 kHz MAS frequency at 278 (K). Alanine Cα–Cβ and proline Cδ–Cβ correlations with chemical shift-dependent secondary structure are marked. FT-IR spectra of (D) TDP-13, (E) TDP-11, and (F) TDP-10 aggregates in the amide I and II range, displaying the experimental (black) and fitted curve (red). The deconvolution of amide bands are shown on the FTIR spectra, displaying contribution of secondary structure elements, namely parallel and antiparallel β-sheet: 1,634 cm⁻¹ (dark blue), 1,620 cm⁻¹ (brown), and 1,690 cm⁻¹ (grey); α-helix: 1,658 cm⁻¹ (sky blue); turns: 1,675 cm⁻¹ (yellow), and random coil: 1,646 cm⁻¹ (green).

FIGURE 5

Investigation of the amyloid core of TDP-13 amyloid aggregates. (A) Aliphatic and (B) aromatic region of 2D¹³C–¹³C correlation experiment of TDP-13 amyloid aggregates recorded at a ¹H frequency of 800 MHz at 278 K and 11 kHz MAS. A PDSD mixing time of 150 ms was used to favor sequential (i.e., residue i to i ± 1) connectivity indicated by color-coded dotted lines. (C) The secondary chemical shift analysis of stretches 318–322, 333–335, and 367–370 comparing experimentally obtained chemical shift with that of random-coil conformation using the equation ΔδCα–ΔδCβ (Wang and Jardetzky, 2002). Positive and negative values corresponds to α-helical or β-strand conformation, respectively.