Figure 7.

Mutant TDP43-EGFP is cytoplasmically mislocalized, and cytoplasmic TDP43-EGFP is sufficient to produce neurodegeneration. A, The mean NCR of TPD43-EGFP, calculated from the fluorescence intensity in the nucleus and cytoplasm of transfected cells, was lower in cells expressing mutant TDP-43-EGFP (blue, n = 261) than in those expressing WT protein (green, n = 214), consistent with the mislocalization of TDP43(A315T)-EGFP to the cytoplasm (*p = 0.003, Kolmogorov–Smirnov test). Solid lines mark the normal distribution curves for each population, and shaded regions represent the proportions of neurons with the indicated NCR values plotted as a histogram. Virtually all neurons expressing TDP43(mNLS)-EGFP (red, n = 107) had an NCR <1, confirming the predominantly cytoplasmic localization of the protein. NCR distribution curves and histograms were generated from expression data pooled from three separate experiments. B, Schematic of TDP43-EGFP highlighting the NLS encompassing residues 78–84. TDP43(mNLS)-EGFP was created by substituting nonpolar alanine residues (AAA) for the basic residues (KRK) at positions 82–84 of TDP43(WT)-EGFP. C–E, Confocal fluorescence microscopy of neurons expressing TDP43(mNLS)-EGFP, demonstrating cytoplasmic localization of the fusion protein. C, EGFP fluorescence. Scale bar, 10 μm. D, TDP43 immunostaining. E, Merged image with Hoechst nuclear staining in blue and overlap in yellow. F, Kaplan-Meier survival analysis and cumulative risk of death curves for neurons expressing TDP43(WT)-EGFP (n = 998), TDP43(A315T)-EGFP (n = 1106), and TDP43(mNLS)-EGFP (n = 732). Both TDP43(A315T)-EGFP and TDP43(mNLS)-EGFP were highly toxic in transfected cells (*p < 0.0001, log-rank test), but there was no statistically significant difference between the effects of TDP43(A315T)-EGFP and TDP43(mNLS)-EGFP on cellular survival (NS, not significant; p > 0.05, log rank test). Cumulative risk of death plots included data from five independent experiments.