Fig. 10

Impaired NHEJ is present in human ALS associated with TDP-43 M337V mutations. a (top panel) Immunocytochemistry for 53BP1 of TDP-43 M337V fibroblasts derived from ALS pre-symptomatic carriers, an affected ALS patient and control individuals. Confocal microscopy and quantification of the number of 53BP1 foci per cell revealed that significantly more DNA damage was present in ALS and pre-symptomatic fibroblasts compared to controls, and in the symptomatic compared to pre-symptomatic fibroblasts. One-way ANOVA with Tukey correction for multiple comparison. Mean ± SEM, ****p < 0.0001, *p < 0.05. 40 fibroblasts per individual were analyzed. Scale bar = 10 μm (bottom panel) Immunoblotting confirmed enhanced expression of DNA damage markers 53BP1 and γH2AX in TDP-43 M337V pre symptomatic compared to control fibroblasts (b) Fibroblasts from two TDP-43 M337V presymptomatic carriers and two control individuals were co-transfected with digested total NHEJ reporter EJ5GFP and empty mCherry vector via nucleofection. The efficiency of total NHEJ was determined by the % of cells displaying GFP fluorescence within mCherry transfected cells. Quantification of GFP-positive cells indicates significantly less NHEJ repair in TDP-43 M337V fibroblasts compared to controls. One-way ANOVA with Tukey correction for multiple comparison, Mean ± SEM, **p < 0.01, *p < 0.05. Scale bar 10 μm. At least 35 cells per group were analysed, N = 3. c TDP-43 M337V fibroblasts derived from pre-symptomatic carriers display pTDP-43 positive structures in the cytoplasm (white arrow), unlike control fibroblasts. One-way ANOVA with Tukey correction for multiple comparison, Mean ± SEM, ****p < 0.0001. Scale bar 10 μm. At least 82 fibroblasts were analysed within 10 fields per group