Fig 2: A schematic model of the dual functions of CHMP2B in regulating autophagy and TDP-43 phosphorylation. (a) A normal level and function of CHMP2B are required to maintain the autophagy function and suppress ubiquitination and protein turnover of the kinase CK1 that phosphorylates TDP-43. (b) KD of CHMP2B releases the suppression of CK1 ubiquitination, which promotes CK1 turnover, leading to decreased CK1 abundance and reduced TDP-43 phosphorylation levels. Meanwhile, down-regulation of CHMP2B causes autophagy impediment, leading to the cytotoxicity. (c) OE of CHMP2B also impairs the autophagy pathway. At the same time, it enhances the suppression of CK1 ubiquitination, which reduces CK1 degradation, increases CK1 levels, and promotes TDP-43 hyperphosphorylation. In this case, both impaired autophagy and increased TDP-43 phosphorylation contribute to the cytotoxicity. (d) The FTD-3–associated CHMP2BIntron5 is a hypermorphic or dominant mutation that manifests strikingly more severe autophagy dysfunction and cytotoxicity than WT CHMP2B but is a hypomorph with regard to the function of CHMP2B in regulating TDP-43 phosphorylation. Together, CHMP2B plays the dual functions in regulating autophagy and TDP-43 phosphorylation, and the two functions may be executed independently through different mechanisms.

Fig 3: Alternative local conformations of the type A FTLD-TDP filament fold.a, Cryo-EM maps of TDP-43 filaments from type A FTLD-TDP with different local conformations of the N-terminal region (cyan arrows) and of the turn connecting the fourth layer to the fifth (yellow arrows), shown as central slices perpendicular to the helical axis. Scale bars, 25 Å. b, Fourier shell correlation (FSC) curves for the two independently-refined cryo-EM half-maps (black lines); for the refined atomic model against the cryo-EM density map (magenta); for the atomic model shaken and refined using the first half-map against the first half-map (cyan); and for the same atomic model against the second half-map (yellow) . FSC thresholds of 0.143 (black dashed line) and 0.5 (magenta dashed line) are shown. c, Local resolution estimates for the cryo-EM density maps. d, Cryo-EM density maps viewed along the helical axis. Scale bar, 10 Å. e, Cryo-EM density maps and atomic models, shown for a single TDP-43 molecule perpendicular to the helical axis.

Fig 4: CK1 inhibition diminishes CHMP2B-mediated cell death in neuroblastoma N2a cells. (a and b) Representative PI staining images (a) and quantification (b) of the cell death of N2a cells transfected with WT CHMP2B or CHMP2BIntron5. (c–e) OE of WT CHMP2B or CHMP2BIntron5 promotes hyperphosphorylation of endogenous TDP-43 (enTDP-43) in N2a cells, examined by Western blot (c) and quantified in (d and e). (f–h) The CK1 inhibitor D4776 (5 µM, 12 h) potently suppresses phosphorylation of TDP-43-HA in N2a cells. (i and j) Quantification (i) and representative PI staining images (j) show remarkable suppression of CHMP2B-mediated cell death by the CK1 inhibitor D4776 in N2a cells. Vehicle control, DMSO. BF, brightfield. Mean ± SEM, n = ∼500 cells each group of pooled results from three independent repeats (b and i) and n = 3–4 (d, e, g, and h). One-way ANOVA with Tukey's HSD post-hoc test (b, d, e, and i) and two-tailed Student’s t test (g and h); *, P < 0.05; ***, P < 0.001. ns, not significant. Scale bars, 50 µm. Source data are available for this figure: SourceData F5.

Fig 5: Cryo-EM structures of TDP-43 amyloid filaments from individuals with type A FTLD-TDP.a, Cryo-EM maps of TDP-43 filaments from the prefrontal cortex of three individuals with type A FTLD-TDP, shown as central slices perpendicular to the helical axis. GRN indicates individuals with mutations in GRN associated with type A FTLD-TDP. Scale bars, 25 Å. b, Schematic of the domain organization of TDP-43. Disease-associated phosphorylation sites are shown. The black line indicates the region that forms the ordered filament fold. NLS, nuclear localization signal. c, Amino acid sequence alignment of the secondary structure elements of the filament fold. Arrows indicate β-strands. d, Cryo-EM map shown at high (grey) and low (yellow) contour levels and atomic model, shown for a single TDP-43 molecule perpendicular to the helical axis. The five layers of the filament fold are labelled. Less well-resolved protein-like density extending from R272 (black arrow), an isolated peptide-like density adjacent to G351–N355 (yellow arrow), and non-protein densities within cavities between layers 1 to 3 (red arrows) are indicated. In c and d, the glycine-rich (G274–G310, magenta), hydrophobic (M311–S342, white) and Q/N-rich (Q343–Q360, green) regions are highlighted.