S1R activation rescued touch-escape response of TDP43^G348C-expressing zebrafish larvae. (A) Touch-escape response of control larvae or larvae expressing mCherry alone or with TDP43^G348C. Swimming distance in 5 s was measured after tactile stimulation of 5 days post-fertilization (dpf) larvae. (B) Touch-escape response of control larvae or larvae expressing TDP43^G348C, TDP43^G348C + S1R or S1R alone. (C) Touch-escape response of control larvae, larvae expressing TDP43^G348C alone or treated with 5 or 10 μM of PRE-084. (D) Touch-escape response of control larvae or larvae expressing TDP43^G348C alone, treated with PRE-084 (5 μM) or treated with PRE-084 and NE-100 (5 μM each). (E) Touch-escape response of control, TDP43^G348C larvae treated or not with SA4503 (5 μM). In all figures data from 20 were averaged and presented as mean ± SEM. Statistical analysis was performed using ANOVA followed by Tukey's test (*p < 0.05; **p < 0.01; ***p < 0.001).

PRE-084 ameliorated TDP43^G348C-larvae mobility and axonal projection. (A) Mobility of control larvae, larvae expressing TDP43^G348C alone or treated with 5 μM of PRE-084 during the light/dark phases in the VMR test. The mobility was measured during 1 min each. AU: arbitrary unit. (B) Quantitative analyses from (A) showing the relative mobility of controls, larvae expressing TDP43^G348C alone or treated with PRE-084. Data were the mean of the mobility during the two OFF periods minus ON periods (OFF1-ON1; OFF2-ON2). Relative mobility from >100 larvae was expressed as percent of control and presented as mean ± SEM. Statistical analysis was performed using ANOVA followed by Tukey's test (**p < 0.01; ***p < 0.001). (C) Representative photomicrographs showing three axonal projections in the zone of analysis. Scale bar: 50 μm. (D) Quantitative analyses of axonal projections (as shown in (C)) in control larvae or larvae expressing TDP43^G348C alone or treated with PRE-084. Data from 42 to 54 motor neurons were averaged and presented as mean ± SEM. Statistical analysis was performed using ANOVA followed by Tukey's test (*p < 0.05; ***p < 0.001).

PRE-84 boosted maximal mitochondrial respiration of TDP43^G348C-expressing larvae. (A) Oxygen consumption rate (OCR) was measured using the Seahorse system on control and TDP43^G348C larvae. (B) Basal mitochondrial respiration, maximal capacity of mitochondrial respiration and non-mitochondrial respiration were measured on control and TDP43^G348C larvae treated or not with PRE-084. Data from n = 18–33 larvae were presented as mean ± SEM. Statistical analysis was performed by the Student t-test (*p < 0.05; **p < 0.01).

TDP43^G348C increased sensitivity to ER stress. (A) Transcript levels were evaluated from control or TDP43^G348C larvae, treated or not with the ER stress inducer, tunicamycin, for 24 h. Data from n = 5 were presented as mean ± SEM. Statistical analysis was performed by using ANOVA followed by Tukey's test (*p < 0.05, **p < 0.01 and ***p < 0.001 versus control condition; Фp<0.05, ФФp<0.01). (B) As in (A) for UPR gene targets: BiP and CHOP (**p < 0.01 and ***p < 0.001 versus control condition; ФФp<0.01). (C) As in (A) for NRF2 isoforms: NRF2a and NRF2b (*p < 0.05 and ***p < 0.001 versus control condition). (D) Western blot showing BiP and ATP5α protein levels from control or TDP43^G348C larvae treated or not with tunicamycin for 24 h. (E) Densitometric analysis of BiP protein levels in zebrafish larvae from (D). ATP5α was used as a reference to normalize BiP levels. Data from 5 independent samples were averaged and presented as mean ± SEM. Statistical analysis was performed using a Student's t-test (*p < 0.05).

PRE-084 enhanced ER stress response in TDP43^G348C-zebrafish. (A) Transcript levels of UPR genes in TDP43^G348C larvae, treated or not with PRE-084 after an ER stress induction with tunicamycin for 24 h. Data from n = 8–9 were presented as mean ± SEM. Statistical analysis was performed by using Student t-test (*p < 0.05). (B) As in (A) but after an ER stress induction for 2 h. Data from n = 6–7 were presented as mean ± SEM. Statistical analysis was performed by using Student t-test (*p < 0.05; **p < 0.01). (C) Western blot showing BiP and ATP5α protein levels larvae expressing TDP43^G348C treated or not with PRE-084 after 2 h with tunicamycin condition. (D) Quantitative analysis of BiP protein levels in zebrafish larvae from (C). ATP5α was used as a reference to normalize BiP levels. Data from 8 to 9 independent samples were averaged and presented as mean ± SEM Statistical analysis was performed by using Student t-test (*p < 0.05).

PRE-084 boosted NRF2 signalling cascade and alleviates TDP43^G348C toxicity. (A and B) RNA expression levels of NRF2 and its targets: GCLC, GCLM, PRDX6 and PGD in TDP43^G348C larvae treated or not with PRE-084 after an ER stress induction with tunicamycin for 24 h (A) or 2 h (B). Data from n = 8–9 (24 h) and n = 6–7 (2 h) were presented as mean ± SEM. Statistical analysis was performed by the Student t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Increasing NRF2 levels reduced TDP43 toxicity. (A) RNA expression levels of NRF2 in larvae treated or not with sulforaphane. Data from n = 6 were presented as mean ± SEM. Statistical analysis was performed by the Student t-test (**p < 0.01; ***p < 0.001). (B) RNA expression levels of NRF2 targets: GCLC, GCLM, PRDX6 and PGD in larvae treated or not with sulforaphane. Data from n ≥ 5 were presented as mean ± SEM. Statistical analysis was performed by the Student t-test (**p < 0.01; ***p < 0.001). (C) Touch-escape response of control larvae or larvae expressing TDP43^G348C and treated or not with sulforaphane. Data from 25 were averaged and presented as mean ± SEM. Statistical analysis was performed by Tukey's test (*p < 0.05; ***p < 0.001). (D) Touch-escape response of control larvae or larvae expressing TDP43^G348C alone or with NRF2. Data from 25 larvae were averaged and presented as mean ± SEM. Statistical analysis was performed using ANOVA followed by Tukey's test (***p < 0.001).