Schematic of transgene used to create pure repeat RAN transgenic C9orf72-ALS/FTD zebrafish lines. The 2mer control line contains two repeats of GGGGCC (G4C2), the sense (S) line contains 45 repeats of G4C2 and the antisense (AS) line contains 39 repeats of CCCGG (C4G2). A V5 epitope tag is in all 3 frames following the repeats (3 × V5) and transgenes also carry the hsp70-DsRed transgene. No ATG codon exists that could allow the translation of the repeats in all 3 frames. ALS = amyotrophic lateral sclerosis; FTD = frontotemporal dementia; RAN = repeat-associated non-AUG. [Color figure can be viewed at www.annalsofneurology.org]

Western blot of RAN-translated products in transgenic 2mer (Ctrl), sense (S), and antisense (AS) 5 days postfertilization embryonic zebrafish. Three 2mer (Ctrl), 5 sense (S), and 4 antisense (AS) generated transgenic lines were probed with anti-V5, anti-DsRed and anti-tubulin (loading control) antibodies. RAN = repeat-associated non-AUG.

Embryonic and adult C9orf72-ALS/FTD zebrafish lines produce multiple RAN-translated DPR species. V5-DPRs and poly-repeats species GP, GR, and PR in (A) sense (S5) and (B) antisense (AS1) 5 days postfertilization embryonic lysates compared to non-transgenic (NTG) and 2mer (Ctrl). V5-DPR species detected in 12 month (C) brain and (D) muscle lysates of sense (S5) and antisense (AS1) C9orf72-ALS/FTD zebrafish. Arrows indicate DPR bands which are proposed to be driven by RAN-translation. Asterisks indicate non-specific antibody binding. ALS = amyotrophic lateral sclerosis; FTD = frontotemporal dementia; DPR = dipeptide repeat; RAN = repeat-associated non-AUG.

Adult C9orf72-ALS/FTD zebrafish show reduced swimming endurance. Kaplan–Meier analysis of swim tunnel performance in 6-month 12-month-old zebrafish. (A) The 2mer (Ctrl) zebrafish show no significant decline in swimming endurance between 6- and 12-month time points. Both (B) sense (S5) and (C) antisense (AS1) zebrafish lines significantly decline in swimming endurance between 6 and 12 months old. N = 9 2mer (Ctrl), N = 10 sense (S5) and N = 10 antisense (AS1). Log-rank (Mantel-Cox) test; *p < 0.05, **p < 0.01, ***p < 0.001. ALS = amyotrophic lateral sclerosis; FTD = frontotemporal dementia. [Color figure can be viewed at www.annalsofneurology.org]

Hypothermic cooling reduces DPR expression in C9orf72-ALS/FTD cell models. (A) HEK293T, N2A and HeLa cells were transfected with control (C), sense (S), and antisense (AS) constructs and protein lysates were collected following 48 hours of hypothermic cooling (32°C). Western blot analysis and quantification of (B–D) V5-DPRs and (E–G) cold shock proteins CIRBP and RBM3. Data presented as mean ± SD. One-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ALS = amyotrophic lateral sclerosis; ANOVA = analysis of variance; CIRBP = cold-inducible RNA-binding protein; FTD = frontotemporal dementia; DPR = dipeptide repeat; SD = standard deviation. [Color figure can be viewed at www.annalsofneurology.org]

Hypothermic-TTM reduces DPR production in 5 days postfertilization embryonic C9orf72-ALS/FTD zebrafish. Western blot and MSD poly(GP) ELISA analysis of 5 days postfertilization embryonic lysates following 72 hours of hypothermic cooling. (A, B) anti-V5-DPR immunostaining and (C, D) MSD poly(GP) ELISA analysis of sense (S5) embryonic lysates. (E, F) The anti-poly(GP) immunostaining and (G, H) MSD poly(GP) ELISA analysis of antisense (AS1) 5 days postfertilization embryonic lysates. MSD ELISA assay responses were interpolated using a GP (7) standard prior to normalization. Western blot – N = 5 to 6 clutches per treatment group; poly(GP) MSD ELISA – N = 3 clutches per treatment group. Data presented as mean ± SD. Unpaired t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ALS = amyotrophic lateral sclerosis; ELISA = enzyme-linked immunosorbent assay; FTD = frontotemporal dementia; DPR = dipeptide repeat; MSD = Meso Scale Discovery; SD = standard deviation; TTM = therapeutic temperature management. [Color figure can be viewed at www.annalsofneurology.org]