Figure 1. The toxicity of TDP43 depends strongly on dose.

(a, b) Histograms of TDP43(WT)-EGFP and TDP43(A315T)-EGFP levels in individual neurons. Increasing amounts of DNA significantly shifted the distribution of log-transformed expression levels towards higher values (p < 0.01 for all comparisons, two-sided Kolmogorov-Smirnov test). TDP43(WT)-EGFP, n=1287 neurons, with 290–342 cells per group; TDP43(A315T)-EGFP, n=1290, with 315–332 neurons per group. Values pooled from 12 wells per condition, performed in duplicate. (c) Automated fluorescence microscopy. Primary rodent cortical neurons were transfected with mApple and TDP43-EGFP, and survival was determined by repeated imaging at regular intervals. The last time at which the cell was noted to be alive (red arrows) was used as the time of death. Cells that survive the entire length of the experiment (blue arrow) were censored. Scale bar = 25 μm. (d, e) Cumulative risk of death over time for neurons transfected with EGFP, TDP43(WT)-EGFP or TDP43(A315T)-EGFP. Supplementary Table 1 lists the number of neurons, hazard ratios, 95% CI, and p values for each condition, as determined by Cox proportional hazards analysis. Results pooled from 12 wells per condition, performed in duplicate. (f, g) Linear regression of calculated hazard ratio (± SEM) as a function of total TDP43(WT)-EGFP (R² = 0.9353) or TDP43(A315T)-EGFP (R² = 0.9574) level. The total TDP43 level was determined by quantitative immunocytochemistry using TDP43-specific antibodies (Supplementary Fig. 3) and normalized to the amount of TDP43 in non-transfected neurons. Dotted lines, reference (HR = 1).