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

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 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

Study design.

<p>For each step, the number of resulting SNP-transcript pairs in <i>cis</i> is shown.</p

eQTLs with a nominally significant GWAS p value in the replication data.

<p>Independent eQTLs are based on LD-based SNP clumping. For each locus, the clump index SNP (with the lowest p value) is shown. For the GWAS replication results, Bonferroni corrected p values are given for the testing of 322 clumps. SNP association results in the joint GWAS data were based on a total of 3,568 ALS patients and 10,163 controls. For the eQTL direction of effect, ‘+’ means the SNP minor allele was associated with increased expression levels, ‘–’ means decreased gene expression. Chr, chromosome; LD, linkage disequilibrium; GWAS, genome-wide association study; OR, odds ratio; p bonf., Bonferroni corrected p value; n.s., not significant; eQTL, expression quantitative trait locus.</p

Results for fine-mapping of loci previously associated with ALS.

<p>The minor allele of rs10122902 was associated with increased <i>C9orf72</i> expression levels, while the minor allele of rs1565948 was associated with decreased expression. LD estimates with SNP rs3849942 and SNP association results in the joint GWAS data were based on a total of 3,568 ALS patients and 10,163 controls. The expression explained variance (R²) was estimated from expression data from both discovery and replication eQTL datasets combined. <i>C9orf72</i>, chromosome 9 open reading frame 72; Chr., chromosome; LD, linkage disequilibrium; GWAS, genome-wide association study; OR, odds ratio; eQTL, expression quantitative trait locus.</p

Regional linkage disequilibrium (LD) near the <i>CYP27A1</i> locus on chromosome 2.

<p>Top: the position of GWAS SNPs and RefSeq genes located within the regional LD block are drawn. On the <i>X</i>-axis, genomic position in kb, aligned to NCBI genome build 36 coordinates. On the left <i>Y</i>-axis, −log₁₀(p values) for the strongest <i>cis</i> eQTL association for a gene in the replication data, the vertical position of genes (drawn as arrows) are aligned to this axis and thus represent statistical significance. For one gene (<i>RQCD1</i>), no SNP-transcript pair and, therefore, no eQTL p value was available in our data. This gene is shown as a dashed arrow. On the right <i>Y</i>-axis, −log₁₀(p values) from the replication GWAS analysis for SNPs within the region (black line), SNPs modulating <i>CYP27A1</i> expression are shown as black dots, other SNPs are grey. Bottom: pairwise linkage disequilibrium for HapMap phase III release 2 SNPs (CEU+TSI populations). The LD plot was created in Haploview v4.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035333#pone.0035333-Barrett2" target="_blank">[50]</a>, using the standard <i>D'</i>/LOD color scheme.</p

Data_Sheet_1_Telomere length analysis in amyotrophic lateral sclerosis using large-scale whole genome sequence data.pdf

BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons, leading to progressive weakness of voluntary muscles, with death following from neuromuscular respiratory failure, typically within 3 to 5 years. There is a strong genetic contribution to ALS risk. In 10% or more, a family history of ALS or frontotemporal dementia is obtained, and the Mendelian genes responsible for ALS in such families have now been identified in about 50% of cases. Only about 14% of apparently sporadic ALS is explained by known genetic variation, suggesting that other forms of genetic variation are important. Telomeres maintain DNA integrity during cellular replication, differ between sexes, and shorten naturally with age. Sex and age are risk factors for ALS and we therefore investigated telomere length in ALS.MethodsSamples were from Project MinE, an international ALS whole genome sequencing consortium that includes phenotype data. For validation we used donated brain samples from motor cortex from people with ALS and controls. Ancestry and relatedness were evaluated by principal components analysis and relationship matrices of DNA microarray data. Whole genome sequence data were from Illumina HiSeq platforms and aligned using the Isaac pipeline. TelSeq was used to quantify telomere length using whole genome sequence data. We tested the association of telomere length with ALS and ALS survival using Cox regression.ResultsThere were 6,580 whole genome sequences, reducing to 6,195 samples (4,315 from people with ALS and 1,880 controls) after quality control, and 159 brain samples (106 ALS, 53 controls). Accounting for age and sex, there was a 20% (95% CI 14%, 25%) increase of telomere length in people with ALS compared to controls (p = 1.1 × 10−12), validated in the brain samples (p = 0.03). Those with shorter telomeres had a 10% increase in median survival (p = 5.0×10−7). Although there was no difference in telomere length between sporadic ALS and familial ALS (p=0.64), telomere length in 334 people with ALS due to expanded C9orf72 repeats was shorter than in those without expanded C9orf72 repeats (p = 5.0×10−4).DiscussionAlthough telomeres shorten with age, longer telomeres are a risk factor for ALS and worsen prognosis. Longer telomeres are associated with ALS.</p