TDP-43 localization in zebrafish embryos.

<p>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.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013368#pone.0013368-Lemmens2" target="_blank">[25]</a>. 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.</p

Overexpression of human PGRN mRNA prevents the decrease in axon outgrowth produced by knockdown of zebrafish PGRN.

<p>A) The decreased axonal length produced by knockdown of <i>grna</i> with a 5′UTR MO was rescued by co-expression of PGRN mRNA. * significantly different from grna CO MO, p<0.02; ̂ significantly different from <i>grna</i> MO + GFP, p<0.0001. Bars represent mean ± s.e.m. Buffer injected: n = 20, CO-MO: n = 15, CO-MO + PGRN: n = 15, grna MO: n = 15, grna MO + GFP: n = 15, grna MO + PGRN: n = 13. B) Real time PCR analysis of cDNA reverse transcribed from RNA extracted from either non-injected or PGRN mRNA injected zebrafish embryos confirmed the presence of human PGRN mRNA in injected embryos (*p<0.0001). C) Quantification of human PGRN protein levels in non-injected and PGRN mRNA injected zebrafish embryos (24hpf) by ELISA confirmed the overexpression of human PGRN protein following PGRN mRNA injection (100 and 250 ng/µl). *Significantly different from non-injected, p<0.043 (post-hoc Wilcoxon Signed Ranks test) following a significant Friedman Test, p = 0.015.</p

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

<p>A) Anti-V5 immunoblot confirmed similar expression levels of Wt and Mt TDP-43 following TDP-43 mRNA injection. Protein produced by <i>in vitro</i> 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).</p

PGRN knockdown results in reduced motor axon outgrowth.

<p>A) Knockdown of <i>grna</i>, by morpholino targeted to both the start codon (ATG) and 5′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 <i>grnb</i> 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.</p

Punctuate staining with SG marker eIF3e was commonly found adjacent to or surrounding FUS-GFP SGs.

<p>Scale bars  = 10 µm; Insets  = 1 µm.</p

Whole mount and cell cultures of FUS-GFP transgenic zebrafish.

<p>(<b>A</b>) Transgenic zebrafish larvae whole mounts showed cytosolic mislocalization of mutant human FUS in FUS-R521C-GFP in comparison to FUS-WT-GFP which was restricted to cell nuclei. (<b>B</b>) FUS-R521C-GFP showed greater cytosolic distribution in comparison to FUS-WT-GFP in zebrafish primary cell cultures. (C) Confocal images of 48 hpf transgenic zebrafish spinal cord further demonstrate mislocalization of mutant FUS-521C-GFP (green) in motor neurons (red) (arrows). Images are maximum projections captured using a Leica SPE5 confocal microscope. Sagittal sections (upper images) of Tg(s1020tGAL4: UASmCherry) (Scott and Baier, 2009) and transverse sections (lower images) of Tg(HB9: mK02caax) membrane localised mk02 expressed in motorneurons by HB9 promoter (Flanagan-Steet et al 2005) with either FUS-WT-GFP or FUS-521C-GFP as indicated. Scale bar  = 20 µm.</p

Quantification of SG assembly and reversibility

<p>(<b>A</b>) FUS-R521C-GFP generated SGs in almost double the number of cells compared to FUS-WT-GFP after 40 minutes heat shock. Further, FUS-R521C-GFP cells were less able to reverse SGs compared to FUS-WT-GFP cells. (<b>B</b>) Quantification of the number of SGs per cell in SG containing cells after heat shock and recovery in three experiments showed that FUS-WT-GFP generated ∼5 FUS-containing SGs per cell and recovered to ∼2 SGs per cell. By contrast, FUS-R521C-GFP SG-containing cells remained at ∼12 SGs per cell before and after heat shock recovery despite recovery of many surrounding cells. *P≤0.05 and ***P≤0.005. Error bars indicate SE.</p

Ubiquitous FUS-GFP SG assembly in zebrafish cells.

<p>(<b>A</b>) FUS-GFP SGs formed in cultured transgenic zebrafish cells after heat-shock at 40°C for 30 mins (arrows). However, SGs were more abundant in mutant FUS-R521C-GFP cultures (right panels) than in FUS-WT-GFP. (<b>B</b>) Motor neurons (39.4D5-labeled cells) were not particularly susceptible to SG assembly. (<b>C</b>) SGs were reversible when cells were allowed to recover at 37°C for another 30 mins. Some persistent SGs were still present particularly in FUS-R521C-GFP cultures. Motor neurons labeled with 39.4D5 readily reversed SGs. (<b>D</b>) Stress granules (SGs) were also induced by sodium arsenite (Na₃AsO₃; 0.2 mM) treatment. SGs formed in mutant (arrows) but not in the FUS-WT-GFP line after Na₃AsO₃ treatment for 1 hr. Similar to heat-shocked cells, 39.4D5-labeled cells were no more susceptible to chemical-induced SG formation. (<b>E</b>) Chemical-induced FUS-GFP containing SGs were reversible in both lines. Reversibility also occurred readily in 39.4D5 labelled motor neurons.</p

Mislocalization of mutant FUS-GFP was also found in motor neurons.

<p>(<b>A</b>) FUS-R521C-GFP was similarly mislocalized to the cytosol in motor neurons (labeled with 39.4D5 for islet1 and islet2 homeodomain marker). (<b>B</b>) Quantification of FUS-GFP signal in nucleus vs. cytosol in 39.4D5 marked motor neurons demonstrated that FUS-R521C-GFP was significantly more cytosolic compared to FUS-WT-GFP (post-hoc Tukey HSD. ** = P<0.01, N.S. = >0.05, n = 40 for all samples. Error bars represent SE. Scale bars  = 20 µm.</p