Injection of ATG poly-GR evokes apoptosis and aberrant motor neuron axon morphology in 1-4-dpf zebrafish embryos. (A) Maximum projection of z-stack images of the SecA5 fluorescent reporter line (green) embryos 48 h after injection with 10 pg ATG poly-GR, 10 pg TAG poly-GR or 400 pg mCherry only. (B) Quantification of z-stack images of the SecA5 fluorescent reporter line embryos after injection with 10 pg ATG poly-GR, 10 pg TAG poly-GR or 400 pg mCherry only at 1-4 dpf. N=minimum of ten fish per group (mean±s.e.m.). P<0.0001 (Kruskal–Wallis test). *P<0.05, ***P<0.0001 (Dunn's post-hoc multiple comparison test), differences of ATG poly-GR-injected fish compared to 10 pg TAG poly-GR and mCherry only at 1-4 dpf. (C) Synaptic vesicle (SV2) in combination with α-bungarotoxin (BTX) staining demonstrates aberrant axonal protrusions in the tail of 2-dpf wild-type AB embryos after injection with 10 pg ATG poly-GR compared to 10 pg TAG poly-GR and 400 pg mCherry only. n=10 per group (mean±s.e.m.). (D) Fluorescence of SV2 staining was measured for five fish/group and three neurites per fish, and was significantly reduced in 10 pg ATG poly-GR-injected embryos. P<0.0001 (one-tailed t-test with Welch's correction). Scale bars: 100 µm.

Poly-GR peptides are detected as (peri)nuclear puncta in zebrafish embryos at 1-4 dpf. (A) Immunofluorescence staining for poly-GR (red) in 2 dpf old wild-type AB embryos after injection with 10 pg poly-GR or 400 pg mCherry only. N=30 per group. C9FTD patient frontal cortex sections were used as a positive control. Poly-GR peptides were detected as nuclear or perinuclear dots in all poly-GR-injected fish (arrows). Nuclei were stained with Hoechst. Scale bars: 20 µm. (B) Poly-GR is detected in postmortem frozen brain samples of C9ORF72 FTD/ALS cases (n=7) but not in postmortem frozen brain samples of FTD patients with mutations in progranulin (GRN; n=2), Valosin-containing protein (VCP; n=2), microtubule-associated protein tau (MAPT, n=1) or in brain samples of non-demented controls (n=5) (mean±s.e.m.). One-way ANOVA (P=0.0024) with post Tukey's test indicating a difference between the C9ORF72 FTD/ALS group and all other groups. (C) ELISA for the detection of poly-GR shows a signal of 10-20 pg peptide in 1-4 dpf wild-type AB embryos injected with 10 pg RNA encoding poly-GR. Two-way ANOVA (P=0.0003) with post Bonferroni test indicating that all timepoints were significantly different from mCherry-only-injected fish. n=90 fish per group per timepoint divided over three independent experiments (30 fish per group×three experiments per timepoint). *P<0.05, ***P<0.0001.

Trolox reduces the number of apoptotic clusters and oxidative stress in zebrafish embryos injected with 10 pg poly-GR. (A) Upper panel: maximum projection of z-stack images of the SecA5 fluorescent reporter line embryos 48 h after injection with 10 pg poly-GR and treated with 50 µM Trolox or DMSO only. Lower panel: maximum projection of TUNEL staining of the same treatment groups in wild-type AB fish at 2 dpf. (B) Quantification of SecA5 z-stack images (mean±s.e.m.). N=30 fish per group per day. Kruskal–Wallis test (P<0.0001). Dunn's post-hoc multiple comparison test (***P<0.0003). (C) MitoSOX red staining in 2 dpf wild-type AB embryos after injection with 10 pg poly-GR or 400 pg mCherry only, and treated with DMSO only or Trolox (dissolved in DMSO). MitoSOX signal (magenta) is seen as clusters in the brain and in the spinal cord in the tail. MitoSOX staining also shows ROS reactivity in pigmented cells (arrowheads) and along the tail and head (arrows). (D) MitoSOX signal is significantly higher in 10 pg poly-GR compared to mCherry-only-injected zebrafish embryo tails at 2 dpf. N=8 fish per group (mean±s.e.m.). Two-tailed unpaired Student's t-test (P=0.0002). F-test for equal variances (P=0.914) demonstrated that variances were not significantly different. (E) MitoSOX signal is significantly reduced in 10 pg poly-GR-injected zebrafish embryo tails treated with Trolox versus DMSO only at 2 dpf. N=9 fish per group (mean±s.e.m.). F-test (P=0.045) demonstrated that variances were significantly different. Two-tailed unpaired Student's t-test with Welch's correction P=0.01. H₂O₂-treated embryos used for MitoSOX positive control experiments, with an additional n=8 fish per group, are shown in Fig. S4. **P<0.001, ***P<0.0001. Scale bars: 100 µm.