Figure 2.
TDP-43 Causes mtDNA Release into the Cytoplasm
(A) TDP-43-EGFP (WT or the ALS mutants A315T and Q331K) and FLAG-cGAS were transiently overexpressed in HEK293T cells, followed by extraction of DNA from FLAG immunoprecipitants. Direct qPCR reveals the presence of mtDNA (MT-ND1 and MT-ND2) but not DNA corresponding to abundant nuclear LINE1 elements (L1ORF1 and L1ORF2) or a ribosomal gene (RNA18S) bound to FLAG-cGAS.
(B) Human THP-1 cells with inducible TDP-43 (WT or Q331K) were depleted of mtDNA using EtBr (ρ0). 72 h after TDP-43 induction, IFNB1 and TNF expression was diminished compared with the untreated (UT) control.
(C) TDP-43 overexpressing THP-1 cells as in (B) were subjected to western blot analysis of inflammatory signaling pathways related to type I IFN and NF-κB. Arrows indicate a cut in the membrane to facilitate multiple protein probing. See the Key Resources Table for uncropped blots.
(D) OMX-SR microscopy reveals that TDP-43 (FLAG-tagged, red) translocates into mitochondria (TIM44, blue; TOM20, cyan) and induces relocation of DNA (anti-DNA, green) into the cytoplasm of TDP-43-overexpressing MEFs (scale bars, 5 μm). Overview images are maximum-intensity projections, and magnified images are 3D surface reconstructions using Imaris software (bottom right) (scale bars, 0.5 μm). See also Video S1.
(E and F) Spatial quantification by Imaris software for (E) the percentage of FLAG-TDP-43 in mitochondria (TIM44) and (F) the percentage of DNA outside of mitochondria; 30–40 cells per group.
Data are mean – SEM, pooled from 3 independent experiments ([A], [B], [E], and [F]) or representative of 3 independent experiments ([C] and [D]). The p values were calculated using two-way ANOVA to control in (A) and (B) or unpaired t test in (E) and (F). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figure S2.