Two FTD-ALS genes converge on the endosomal pathway to induce TDP-43 pathology and degeneration

Abstract

Frontotemporal dementia and amyotrophic lateral sclerosis (FTD-ALS) are associated with both a repeat expansion in the C9orf72 gene and mutations in the TANK-binding kinase 1 (TBK1) gene. We found that TBK1 is phosphorylated in response to C9orf72 poly(Gly-Ala) [poly(GA)] aggregation and sequestered into inclusions, which leads to a loss of TBK1 activity and contributes to neurodegeneration. When we reduced TBK1 activity using a TBK1-R228H (Arg²²⁸→His) mutation in mice, poly(GA)-induced phenotypes were exacerbated. These phenotypes included an increase in TAR DNA binding protein 43 (TDP-43) pathology and the accumulation of defective endosomes in poly(GA)-positive neurons. Inhibiting the endosomal pathway induced TDP-43 aggregation, which highlights the importance of this pathway and TBK1 activity in pathogenesis. This interplay between C9orf72, TBK1, and TDP-43 connects three different facets of FTD-ALS into one coherent pathway.

Fig. 1.. pTBK1 accumulates into DPR aggregates in c9FTD-ALS.

(A and B) Immunoblot (A) and quantification (B) of 6-month-old mouse cortical lysates (N = 4 mice). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; A.U., arbitrary units. (C and D) Images (C) and quantification (D) of pTBK1 in 6-month-old mouse cortex (N = 9). Scale bar, 20 μm. (E) Immunofluorescence in 6-month-old mouse cortices. Scale bar, 10 μm. (F and G) Immunoblot (F) and quantification (G) of HEK293T lysates (N = 4 independent experiments). GFP monomer: ~27k Da; GFP-DPRs: high molecular weight (HMW) smears. (H) Immunostaining in the cortex of 3.5-month-old GFP and (GA)₁₀₀-V5 mice and 2-week-old GFP-(GR)₂₀₀ mice. Scale bars, 20 μm. (I) Immunostaining in healthy and C9-FTD frontal cortex (images labeled #1 and #2; scale bar, 10 μm) and hippocampus (images labeled #3; scale bar, 50 μm). (J) Immunofluorescence in healthy and C9-FTD frontal cortex. Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. Data are means ± standard deviations (SDs). *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001; ns, nonsignificant. Statistics by Student’s t tests in (B) and (D) and one-way analysis of variance (ANOVA) in (G). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; G, Gly; P, Pro; R, Arg.

Fig. 2.. Poly(GA) aggregation sequesters TBK1 into inclusions, which limits its activity.

(A and B) Immunoblot (A) and quantification (B) of HEK293T lysates (N = 3 independent experiments). The number symbol in (A) indicates nonspecific bands. (C) Immunofluorescence of HEK293T cells. Scale bar, 10 μm. (D and E) Immunoblot (D) and quantification € of HEK293T lysate fractions (N = 3). S, soluble; Ins, insoluble. The asterisk in (D) indicates higher exposure. (F) Coimmunoprecipitation analysis of HEK293T lysates. P, immunoprecipitation. (G and H) Immunoblot (G) and quantification (H) of HEK293T lysates (N = 3). In all blots, GFP monomer: ~27 kDa; GFP-DPRs: ~50 kDa unaggregated, HMW oligomerized. Data are means ± SDs. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001; ns, nonsignificant. Statistics by one-way ANOVA.

Fig. 3.. Reducing TBK1 activity exacerbates the behavioral, neurodegenerative, and pathological effects of poly(GA) in mice.

(A) Hindlimb clasping scores (N = 9, 10, 16, and 14 mice at each time point). WT, wild-type. (B) Hanging wire test in 3-month-old mice (N = 9, 10, 16, and 14). (C) Whole mouse brain weight (N = 12, 15, 16, and 14). (D and E) Images (D) and quantification (E) of NeuN in mouse cortex (N = 11, 12, 13, and 12). Scale bar, 200 µm. (F and H) Images (F) and quantification (H) of pTBK1 in mouse cortex (N = 14 each). Scale bar, 20 µm. (G and I) Images (G) and quantification (I) of poly(GA) in mouse cortex (N = 14 and 16). Scale bar, 20 µm. (J to L) Immunofluorescence (J) and quantification [(K) and (L)] in mouse cortex (N = 6 each). Scale bar, 10 μm. NT, non-transduced or poly(GA)-negative. Except in (A) and (B), mice were 3.5 months old. Data are means ± SDs. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, nonsignificant. Statistics by two-way ANOVA in (A), Student’s t test in (H) to (I), and one-way ANOVA in remaining panels.

Fig. 4.. Poly(GA) induces endosomal defects that trigger TDP-43 proteinopathy, and a reduction in TBK1 activity intensifies this process.

(A and B) Images (A) and quantification (B) of early endosome antigen 1 (EEA1) in mouse cortex (N = 6, 6, 13, and 13 mice). Scale bar, 20 µm. (C and D) Immunofluorescence (C) and quantification (D) in mouse cortex (N = 6 each). Scale bar, 10 μm. (E and F) Images (E) and quantification (F) of pTDP-43 in mouse cortex (N = 6, 6, 14, and 13). Scale bar, 20 µm. (G and H) Immunofluorescence (G) and quantification (H) in mouse cortex (N = 6 each). Scale bar, 10 μm. (I to K) Immunofluorescence (I) and quantification [(J) and (K)] in HEK293T cells. Scale bar, 10 μm. (L to M) Immunofluorescence (L) and quantification [(M) and (N)] in HEK293T cells ± apilimod treatment (N = 3 independent experiments). Scale bar, 10 μm. All mice were 3.5 months old. Data are means ± SDs. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001; ns, nonsignificant. Statistics by Student’s t test in (J), (K), (M), and (N) and by one-way ANOVA in remaining panels.