ALS post-mortem motor cortex exhibits TUBA4A downregulation. (a) Western blot on SDS-soluble lysates derived from the motor cortex of control (n = 5) and ALS (n = 5) cases using an antibody against TUBA4A (C-term). GAPDH was used as a loading control. (b) Quantification of the expression of TUBA4A as a ratio to GAPDH in the motor cortex. Unpaired t-test. (c) Immunohistochemical staining of the motor cortex of a representative ALS and control case with an antibody against TUBA4A (C-term). Scale bar represents 50 μm. **p < 0.01.

Specific tuba8l2 knockdown in zebrafish induces axonal abnormalities. (a) An ATG morpholino was designed against the Danio rerio TUBA4A orthologue tuba8l2. (b–e) Western blot was performed at 48 hpf after injection of different doses of ATG morpholino against tuba8l2 (0.160 mM, 0.125 mM and 0.050 mM) as well as the injection of a control morpholino (0.160 mM). N = 3 experiments; n = 10–15 zebrafish per group per experiment. Quantification of tuba8l2 panel (c) and α-tubulin panel (e) protein levels relative to GAPDH for the different injection conditions. (f,g) Visualization of motor axons by SV2 immunohistochemistry at 30 hpf after injection of different doses of ATG morpholino against tuba8l2 (0.160 mM, 0.125 mM and 0.050 mM) or a control morpholino (0.160 mM). A non-injected condition was also included. Scale bar represents 50 μm p < 0.0001 (0.160 mM versus AMO control), p < 0.0001 (0.125 mM versus AMO control) and p < 0.0450 (0.050 mM versus AMO control); one-way ANOVA with Dunnett’s multiple comparisons. Axonal length was measured for N = 3 experiments; n = 10–15 zebrafish embryos per group per experiment; with every data point representing the average length of the five measured axons for each zebrafish embryo. *p < 0.05; ****p < 0.0001. AMO, morpholino; hpf, hours post fertilization.

Zebrafish motor behavior deficits are induced by tuba8l2 knockdown. Zebrafish were subjected to a touch-evoked escape response (TEER) assay at 48 hpf after injection of different doses of ATG morpholino against tuba8l2 (0.160 mM, 0.125 mM, 0.050 mM), or a control morpholino (0.160 mM). In addition, non-injected embryos were included in the analysis. (a) Total distance for 0.160 mM (p < 0.0001), 0.125 mM (p = 0.0001) and 0.050 mM (p = 0.1936) compared to AMO control condition. (b) Average velocity for 0.160 mM (p < 0.0001), 0.125 mM (p < 0.0001) and 0.050 mM (p = 0.9814) compared to AMO control condition. (c) Maximal instant velocity 0.160 mM (p < 0.0001), 0.125 mM (p < 0.0001) and 0.050 mM (p = 0.9983) compared to AMO control condition. Kruskal-Wallis test with Dunn’s multiple comparisons panel (a) or one-way ANOVA with Dunnett’s multiple comparisons panels (b,c); N = 3 experiments; n = 10–15 zebrafish embryos per group per experiment; with each data point representing an individual zebrafish embryo. (d) Visual example of the tracking of an escape response in the AMO control condition using the TEER assay in zebrafish embryos at 48 hpf. ***p < 0.001; ****p < 0.0001. AMO, morpholino; hpf, hours post fertilization.

Rescue of axonal length defects by the addition of human wild-type TUBA4A mRNA. Zebrafish embryos were injected with an ATG morpholino against zebrafish tuba8l2 (0.160 mM) with or without the injection of human wild-type TUBA4A mRNA (300 ng/μl). Non-injected embryos were also included in the analysis. (a) Representative whole body images of zebrafish embryos for the different conditions at 48 hpf (scale bar represents 500 μm). (b) At 30 hpf, axonal length was measured for all conditions, with p = 0.0051 (AMO 0.160 mM versus AMO 0.160 mM + TUBA4A mRNA), p < 0.0001 (AMO 0.160 mM versus non-injected) and p < 0.0001 (non-injected versus AMO 0.160 mM + TUBA4A mRNA); one-way ANOVA with Dunnett’s multiple comparisons. Axonal length was measured for N = 3 experiments; n = 10–15 zebrafish per group per experiment; with every data point representing the average length of the five measured axons for each zebrafish embryo. **p < 0.01; ****p < 0.0001. AMO, morpholino; hpf, hours post fertilization.

Rescue of motor behavior deficits by the addition of human wild-type TUBA4A mRNA. Zebrafish embryos were subjected to a touch-evoked escape response (TEER) assay at 48 hpf after injection with an ATG morpholino against zebrafish tuba8l2 (0.160 mM) with or without the injection of wild-type human TUBA4A mRNA (300 ng/μl). Non-injected embryos were also included in the analysis. (a) Total distance for AMO 0.160 mM + TUBA4A mRNA compared to non-injected (non-significant) and AMO 0.160 mM condition (p < 0.0001), and non-injected (p < 0.0001) compared to AMO 0.160 mM condition. (b) Average velocity for AMO 0.160 mM + TUBA4A mRNA compared to non-injected (p = 0.0018) and AMO 0.160 mM condition (p < 0.0001), and non-injected (p < 0.0001) compared to AMO 0.160 mM condition. (c) Maximal instant velocity for AMO 0.160 mM + TUBA4A mRNA compared to non-injected (p = 0.0110) and AMO 0.160 mM condition (p < 0.0001), and non-injected (p < 0.0001) compared to AMO 0.160 mM condition. Kruskal-Wallis test with Dunn’s multiple comparisons; N = 3 experiments; n = 10–15 zebrafish embryos per group per experiment; which each data point representing an individual zebrafish embryo. (d) Representative escape traces from 10 zebrafish embryos per group shown as a visual example. *p < 0.05, **p < 0.01, and ****p < 0.0001. AMO, morpholino; hpf, hours post fertilization.

Zebrafish microtubule polymerization is not affected by tuba8l2 knockdown. At 48 hpf, an EB3 comet assay was performed in single CaP motor neurons after co-injection of an ATG morpholino against tuba8l2 (0.160 mM) or a control morpholino (0.160 mM). (a–d) Quantification of the comet run metrics extracted from kymograms show no difference in microtubule growth kinetics. (a) Average comet distance for AMO 0.160 mM (p = 0.8230) compared to AMO control condition. (b) Average comet duration for AMO 0.160 mM (p = 0.7340) compared to AMO control condition. (c) Average comet velocity for AMO 0.160 mM (p = 0.4605) compared to AMO control condition. (d) Average comet density for AMO 0.160 mM (p = 0.3741). Unpaired t-test (a) or Mann-Whitney test (b,c,d); N = 3 experiments; n = 20–37 zebrafish embryos with each data point representing an individual zebrafish embryo. (e) Representative image (composite z-stack projection) of EB3-GFP in AMO control zebrafish embryo. Red box: representative area for the comet analysis in a distal segment in the CaP. (f) Representative kymograms represent microtubule growth extracted through time-lapse imaging (500 ms/5 min) of EB3-GFP comets in single CaP motor neurons distal arbors at 48 hpf. Each pixel on the Y-axis represents one timepoint image (time) projected against the neurite length (distance) on the X-axis. AMO, morpholino; hpf, hours post fertilization; CaP, caudal primary; scale bar, 25 μm; t, time (5 min); d, distance (average: 17 μm segments).

Specific tuba8l2 knockdown in zebrafish induces changes in post-translational modifications of tubulin. (a–f) Western blot was performed on whole fish lysate from 48 hpf embryos injected with ATG morpholino against tuba8l2 (0.160 mM) vs. control morpholino (0.160 mM). N = 6 experiments; n = 10–30 zebrafish per group per experiment. (a–c) Representative western blot for acetylated α-tubulin (a), detyrosinated α-tubulin (b) and polyglutamylated tubulin (c). (d–f) Quantification of acetylated α-tubulin (d) (p = 0017), detyrosinated α-tubulin (e) (p < 0.0001) and polyglutamylated tubulin (f) (p = 0.2014) protein levels relative to α-tubulin after normalization to GAPDH for the different injection conditions. Data represent mean ± SEM. Unpaired t-test: ** p < 0.01, *** p < 0.001. AMO, morpholino; hpf, hours post fertilization; ctrl, control.