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

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

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

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

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

Data_Sheet_1_TUBA4A downregulation as observed in ALS post-mortem motor cortex causes ALS-related abnormalities in zebrafish.PDF

Disease-associated variants of TUBA4A (alpha-tubulin 4A) have recently been identified in familial ALS. Interestingly, a downregulation of TUBA4A protein expression was observed in familial as well as sporadic ALS brain tissue. To investigate whether a decreased TUBA4A expression could be a driving factor in ALS pathogenesis, we assessed whether TUBA4A knockdown in zebrafish could recapitulate an ALS-like phenotype. For this, we injected an antisense oligonucleotide morpholino in zebrafish embryos targeting the zebrafish TUBA4A orthologue. An antibody against synaptic vesicle 2 was used to visualize motor axons in the spinal cord, allowing the analysis of embryonic ventral root projections. Motor behavior was assessed using the touch-evoked escape response. In post-mortem ALS motor cortex, we observed reduced TUBA4A levels. The knockdown of the zebrafish TUBA4A orthologue induced a motor axonopathy and a significantly disturbed motor behavior. Both phenotypes were dose-dependent and could be rescued by the addition of human wild-type TUBA4A mRNA. Thus, TUBA4A downregulation as observed in ALS post-mortem motor cortex could be modeled in zebrafish and induced a motor axonopathy and motor behavior defects reflecting a motor neuron disease phenotype, as previously described in embryonic zebrafish models of ALS. The rescue with human wild-type TUBA4A mRNA suggests functional conservation and strengthens the causal relation between TUBA4A protein levels and phenotype severity. Furthermore, the loss of TUBA4A induces significant changes in post-translational modifications of tubulin, such as acetylation, detyrosination and polyglutamylation. Our data unveil an important role for TUBA4A in ALS pathogenesis, and extend the relevance of TUBA4A to the majority of ALS patients, in addition to cases bearing TUBA4A mutations.</p

Prevalence of metabolic syndrome and its components in CIAP patients and controls.

<p>Prevalence of metabolic syndrome and its components in CIAP patients and controls.</p

Distribution of 1-deoxySLs in plasma of CIAP-patients and controls.

<p>A significant elevation of 1-deoxySO (A) and 1-deoxySA (B) in plasma of CIAP- or DSPN-patients compared to healthy controls is shown. Not significant (ns): P>0.05; significant: *: P≤ 0.05; **: P≤ 0.01; ***: P≤ 0.001; ****: P≤ 0.0001.</p

Demographic and clinical features in CIAP patients and controls.

<p>Demographic and clinical features in CIAP patients and controls.</p

Plasma sphingolipid profile in CIAP patients and controls.

<p>Plasma sphingolipid profile in CIAP patients and controls.</p