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

Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43) is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTLD). These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation) induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration

A paucigranulocytic asthma host environment promotes the emergence of virulent influenza viral variants

Influenza virus has a high mutation rate, such that within one host different viral variants can emerge. Evidence suggests that influenza virus variants are more prevalent in pregnant and/or obese individuals due to their impaired interferon response. We have recently shown that the non-allergic, paucigranulocytic subtype of asthma is associated with impaired type I interferon production. Here, we seek to address if this is associated with an increased emergence of influenza virus variants. Compared to controls, mice with paucigranulocytic asthma had increased disease severity and an increased emergence of influenza virus variants. Specifically, PB1 mutations exclusively detected in asthmatic mice were associated with increased polymerase activity. Furthermore, asthmatic host-derived virus led to increased disease severity in wild-type mice. Taken together, these data suggest that at least a subset of patients with asthma may be more susceptible to severe influenza and may be a possible source of new influenza virus variants

Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing

Abstract Background Inherent sources of error and bias that affect the quality of sequence data include index hopping and bias towards the reference allele. The impact of these artefacts is likely greater for low-coverage data than for high-coverage data because low-coverage data has scant information and many standard tools for processing sequence data were designed for high-coverage data. With the proliferation of cost-effective low-coverage sequencing, there is a need to understand the impact of these errors and bias on resulting genotype calls from low-coverage sequencing. Results We used a dataset of 26 pigs sequenced both at 2× with multiplexing and at 30× without multiplexing to show that index hopping and bias towards the reference allele due to alignment had little impact on genotype calls. However, pruning of alternative haplotypes supported by a number of reads below a predefined threshold, which is a default and desired step of some variant callers for removing potential sequencing errors in high-coverage data, introduced an unexpected bias towards the reference allele when applied to low-coverage sequence data. This bias reduced best-guess genotype concordance of low-coverage sequence data by 19.0 absolute percentage points. Conclusions We propose a simple pipeline to correct the preferential bias towards the reference allele that can occur during variant discovery and we recommend that users of low-coverage sequence data be wary of unexpected biases that may be produced by bioinformatic tools that were designed for high-coverage sequence data

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