FIGURE SUMMARY
Title

Progranulin is Neurotrophic In Vivo and Protects against a Mutant TDP-43 Induced Axonopathy

Authors
Laird, A.S., Van Hoecke, A., De Muynck, L., Timmers, M., Van Den Bosch, L., Van Damme, P., and Robberecht, W.
Source
Full text @ PLoS One

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.

EXPRESSION / LABELING:
Antibody:
Fish:
Anatomical Term:
Stage: Prim-15
Acknowledgments
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