PGRN knockdown results in reduced motor axon outgrowth.

A) Knockdown of grna, by morpholino targeted to both the start codon (ATG) and 5U′UTR region of zebrafish PGRN sequence, produced a dose dependent decrease in axonal length compared to mismatch Control MO injected embryos.■Significantly different from 200 μM Control MO, p<0.036; # significantly different from 50 μM MO, p<0.001; grna CO-MO (ATG): n = 41; grna CO-MO (UTR): n = 20; grna ATG-MO, 50 μM: n = 40, 100 μM: n = 40, 200 μM: n = 41; grna 5′UTR-MO, 50 μM: n = 27, 100 μM: n = 28, 200 μM: n = 14; Knockdown of grnb produced a similar, but more subtle, axonal shortening. * Significantly different from 600 μM Control MO, p<0.038; # significantly different from 200 μM MO, p<0.05; grnb CO-MO (ATG): n = 27; grnb CO-MO (UTR): n = 10; grnb ATG-MO, 200 μM: n = 40, 400 μM: n = 36, 600 μM: n = 41; grnb 5′UTR-MO, 200 μM: n = 9, 400 μM: n = 12, 600 μM: n = 12; B) The two MO used simultaneously had a cumulative effect; * significantly different from Control MO a + b, p<0.002; # significantly different from all other groups p<0.0001. Buffer injected: n = 20, CO-MO (A + B): n = 34, grnb MO: n = 36, grna MO: n = 36, grna + grnb MO: n = 36. All bars represent mean ± s.e.m.

Overexpression of wild type (Wt) and mutant (Mt) TDP-43 mRNA produces motor axonopathies.

A) Anti-V5 immunoblot confirmed similar expression levels of Wt and Mt TDP-43 following TDP-43 mRNA injection. Protein produced by in vitro translation of the Wt TDP-43 mRNA served as a positive control. B) Overexpression of Wt TDP-43 produced modest axonal shortening (^p<0.001) and aberrant branching (^p<0.001) in comparison with Buffer injection (n = 61). Mt TDP-43 expression (600 ng/μl: n = 37, 650 ng/μl: n = 64, 700 ng/μl: n = 26) produced a more pronounced effect, resulting in significantly shorter axonal lengths (*p<0.001) and more embryos affected by aberrant branching (*p<0.001) than Wt TDP-43 injected embryos (600 ng/μl: n = 35, 650 ng/μl: n = 59, 700 ng/μl: n = 45)

Co-expression of PGRN rescues the axonopathy induced by Mt TDP-43 but not Mt SOD1.

A) Staining of primary motor axons with an anti-synaptic vesicle 2 revealed a decrease in axonal length and increase in aberrant branching in embryos expressing Mt TDP-43 compared with Wt TDP-43. These signs of axonopathy were reduced by co-expression of human PGRN. B) Zebrafish embryos co-expressing Mt TDP-43 and control mRNA (GFP) exhibited decreased axonal outgrowth and increased aberrant branching compared to embryos expressing Wt TDP and GFP (p<0.001 and p = 0.016, respectively). However, co-injection with the equivalent dosage of PGRN mRNA (250 ng/μl) rescued both axopathies described (p<0.043). Mt SOD1 produced motor axon shortening (p<0.001) and increased branching (p<0.001) in comparison with Wt SOD1, as described previously [25]. PGRN co-expression had no significant effect on the Mt SOD1 induced axonopathy but did increase aberrant branching in Wt SOD1 injected embryos (p = 0.006). ^ indicates significantly different from buffer, * significantly different from ‘Wt + GFP’, and # significantly different from ′Mt + GFP′. Bars represent mean ± s.e.m and the number of replicates per group is displayed below each bar.

TDP-43 localization in zebrafish embryos.

Immunofluorescent staining of endogenous zebrafish TDP-43 and overexpressed human TDP-43 was performed in transversely sectioned 30 hpf embryos in order to allow imaging of the spinal cord (shown in schematic diagram, A). TDP-43 localization was nuclear in all embryos examined (B: non injected, C: PGRN MO injected, D: Wt TDP-43 injected, E: Mt TDP-43 (A315T) injected, and F: co-expressing Mt TDP-43 and PGRN). The scale bar indicates a distance of 25 μm. Abbreviations: DSC, Dorsal spinal cord; VSC, Ventral spinal cord; Myo, myotomes; NC, notochord; G, gut.