Wildtype and ALS-linked mutant TDP-43 proteins induce ER stress.

<p>(<b>A</b>) Increase in nuclear CHOP immunoreactivity in Neuro2a cells expressing wildtype and ALS mutant forms of TDP-43 linked with mCherry. (<b>B</b>) Increase in nuclear CHOP immunoreactivity in Neuro2a cells expressing wildtype and ALS mutant forms of TDP-43 linked with EGFP. (<b>C</b>) XBP-1 is activated in mutant TDP-43 Q331K cells, indicating induction of the IRE1 pathway of ER stress. Cells expressing TDP-43 mCherry (first column, red) are shown with XBP-1 (second column, green) and DAPI staining (third column, blue). Merge (fourth column) indicates overlays of the fluorescent images of XBP-1 and DAPI. Arrow indicates cells with increased nuclear XBP-1. Scale bars represent 5 µm. (<b>D</b>) Quantification of transfected cells with activation of XBP-1, as determined by an increase in nuclear XBP-1 immunoreactivity. (<b>E</b>) ATF6 translocates to the Golgi in mutant Q331K cells, indicating activation of the ATF6 pathway of ER stress. Neuro2a cells were co-transfected with mCherry constructs (first column, white) and ATF6-GFP (second column, green). Cells were fixed and immunostained for Golgi marker GM130 (third column, red). Merge (fourth column) indicates overlays of the fluorescent images of ATF6 and GM130. Arrow indicates co-localisation of ATF6 in the Golgi apparatus. Scale bars represent 10 µm. (<b>F</b>) Quantification of transfected cells with activation of ATF6, as determined by co-localisation of ATF6 with GM130. Data are represented as mean ± SEM; *p<0.05 versus mCherry or EGFP controls and #p<0.05 versus respective wildtype TDP-43 proteins, by one-way ANOVA with Tukey's post hoc test.