FIGURE 7.
mFUS motoneurons show signs of increased DNA damage which are potentiated after irradiation. (A) 21 days old motoneurons before and 24 h after irradiation were immunostained by γH2A.X (green) and categorized either negative (I) or positive (II) for DNA damage. (B) Even before γ-irradiation the amount of DNA damage was significantly increased in 21 and 42 days old motoneurons expressing Asp502ThrfS∗27. After irradiation the control as well as the Asp502ThrfS∗27 cell line showed an increased number of γH2A.X+ cells compared to non-irradiated; 42 days old motoneurons expressing mFUS show more DNA damage compared to control. Results are displayed relative to the non-irradiated control line (unpaired t-test; comparison of cell lines was performed by one way ANOVA with Bonferroni post hoc test). (C) The effect of maturation (21 vs. 42 days old motoneurons) on γH2A.X+ cells revealed significant results for control as well as for Asp502ThrfS∗27 (unpaired t-test). (D) 21 days old cells before and 24 h after irradiation were stained for FUS. Without irradiation FUS is predominantly present in the nucleus in control and Asp502ThrfS∗27 cells. Some cytoplasmic FUS staining is seen in young mFUS cells. After irradiation, in control as well as Asp502ThrfS∗27 FUS is mislocalized to the cytoplasm (insert II, red arrowheads) in some motoneurons. In addition to this Asp502ThrfS∗27 motoneurons show large positive FUS inclusions (insert II, red arrowheads). ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001; CNTL, control and mFUS, mutated FUS. Scale bars: 10 μm.