Validation of neurofilament changes in the <i>Dicer</i> cKO mice.

<p>(<b>A</b>) Immunohistochemistry of Nefl in the cortex (a–f) and the dentate gyrus (g–l) of control (a–c, g–i) and <i>Dicer</i> cKO (d–f, j–l) mice. Note the reduction in Nefl signal (in green) in the mutant mice (highlighted in white square). Of mention, changes in the cortex were more pronounced in 13 week-old <i>Dicer</i> cKO mice (shown here) when compared to age-matched controls. Dentate gyrus stainings gave similar results in both 9–10.5 and 13 week-old mice (9.5 week-old mouse shown here). DAPI (nuclei) stainings are shown in blue. Scale bars 20 µm (a–l). (<b>B</b>) Representative (n = 5) western blot analysis of Nefl and Nefh in cortex samples of 9–10.5 week-old control and <i>Dicer</i> cKO mice. Note that only Nefl was downregulated in the <i>Dicer</i> cKO mice. Gapdh was used as internal loading control.</p

Overlapping miRNA targets.

<p>Diagram showing that highly (between 43000 and 165000 reads, left panel) and moderately (between 700 and 6500 reads, right panel) expressed miRNAs have overlapping mRNA targets. Overlapping predicted target genes are annotated.</p

miRNA activity is associated with miRNA abundance.

<p><b>(A, B</b>) Correlation between miRNA quantity and number of seeds with the number of predicted miRNA targets. <i>y</i>-Axis  =  log2(x), <i>x</i>-axis  =  log10(x). <b>(C, D</b>) Correlation between miRNA quantity and number of seeds with the number of validated miRNA targets. For these calculations, we used miRNAs with at least one validated target gene. <i>y</i>-Axis  =  log2(x), <i>x</i>-axis  =  log10(x).</p

Gene Network and Pathway Analysis of Mice with Conditional Ablation of Dicer in Post-Mitotic Neurons

<div><h3>Background</h3><p>The small non-protein-coding microRNAs (miRNAs) have emerged as critical regulators of neuronal differentiation, identity and survival. To date, however, little is known about the genes and molecular networks regulated by neuronal miRNAs <em>in vivo</em>, particularly in the adult mammalian brain.</p> <h3>Methodology/Principal Findings</h3><p>We analyzed whole genome microarrays from mice lacking <em>Dicer</em>, the enzyme responsible for miRNA production, specifically in postnatal forebrain neurons. A total of 755 mRNA transcripts were significantly (P<0.05, FDR<0.25) misregulated in the conditional <em>Dicer</em> knockout mice. Ten genes, including Tnrc6c, Dnmt3a, and Limk1, were validated by real time quantitative RT-PCR. Upregulated transcripts were enriched in nonneuronal genes, which is consistent with previous studies <em>in vitro</em>. Microarray data mining showed that upregulated genes were enriched in biological processes related to gene expression regulation, while downregulated genes were associated with neuronal functions. Molecular pathways associated with neurological disorders, cellular organization and cellular maintenance were altered in the <em>Dicer</em> mutant mice. Numerous miRNA target sites were enriched in the 3′untranslated region (3′UTR) of upregulated genes, the most significant corresponding to the miR-124 seed sequence. Interestingly, our results suggest that, in addition to miR-124, a large fraction of the neuronal miRNome participates, by order of abundance, in coordinated gene expression regulation and neuronal maintenance.</p> <h3>Conclusions/Significance</h3><p>Taken together, these results provide new clues into the role of specific miRNA pathways in the regulation of brain identity and maintenance in adult mice.</p> </div

Gene expression changes in the absence of neuronal Dicer <i>in vivo</i>.

<p>(<b>A</b>) Cluster analysis of microarray data from control and <i>Dicer</i> cKO mice (cortex). Here, all genes significantly changed (P<0.05, FDR <0.25, n = 798) were included in this analysis. Results were generated using Partek Genomics Suite. (<b>B</b>) Histogram showing that 65–75% of misregulated transcripts has less than 1.5-fold difference in gene expression in the <i>Dicer</i> mutant mice when compared to controls. <i>y</i>-Axis  =  log2(x). (<b>C</b>) Validation of selected genes by real-time quantitative RT-PCR. Gapdh was used as normalization control. Statistical significance was determined by a <i>Student unpaired t</i> test (* = p<0.05, ** = p<0.01, *** = p<0.001). Standard deviation is shown.</p

Significant gene networks associated with neuronal miRNA loss in post-mitotic neurons.

<p>(<b>A</b>) Shown here are IPA-generated pathways. Both upregulated and downregulated genes were included in the analysis. Significant biological functions are associated with the regulation of the cytoskeleton. Relationships are primarily due to co-expression, but can also include phosphorylation/dephosphorylation, proteolysis, activation/deactivation, transcription, binding, inhibition, and biochemical modification. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044060#pone.0044060.s001" target="_blank">Figure S1</a> for further details. Nefl downregulation (in blue) was confirmed at the protein level in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044060#pone-0044060-g003" target="_blank">Figure 3</a>. P values were calculated by IPA.</p

IPA networks associated with high-ranking seed sequences.

<p>The top-ranking biological networks associated with (<b>A</b>) miR-124, (<b>B</b>) miR-19, (<b>C</b>) miR-29 and <b>(D</b>) miR-20/17/106/93 predicted target genes are depicted. Related biological functions and P values are indicated and generated using the IPA software. Genes in green are significantly misregulated according to our microarrays. Genes in blue have been validated at the protein level in our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044060#pone.0044060-Hebert1" target="_blank">[14]</a>. Images and P values were generated using the IPA software. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044060#pone.0044060.s001" target="_blank">Figure S1</a> for further details.</p

α-Syn and synphilin-1 equally enhance cell death in aged yeast cells.

<p>A: Quantification of ROS accumulation using DHE staining at different times during growth of yeast strains transformed with an empty plasmid (□, Ctrl.) or constructs allowing for expression of α-Syn (▪), SY^WT (Δ), SY^R621C (▴),α-Syn and SY^WT (○) or α-Syn and SY^R621C (•). B: Quantification of the number of cells that display phosphatidylserine externalization or loss of membrane integrity using annexinV/propidium iodide (PI) co-staining at 36 h of growth in the strains used in A. C: Quantification of viable cells present in the strains used in A at 36 h of growth as determined by their ability to form colonies. D: Fluorescence microscopic visualization of cells expressing combinations of α-Syn, SY^WT or SY^R621C as indicated and stained with DHE (upper panels) or co-stained with annexinV and PI (lower panels) after 36 h of growth. E and F: Quantification of viable cells (E) and cells producing ROS (F) in the strains used in A when kept in culture for two weeks. All data represent mean ± SEM of six independent transformants. Significance of the data was determined by t-tests (* = p<0.05; ** = p<0.01; *** = p<0.001).</p

Co-expression of synphilin-1 increases α-Syn S129-phosphorylation.

<p>A: Phosphorylation of α-Syn at S129 in the BY4741 wild-type strain when the expression of α-Syn was combined with an empty plasmid or constructs allowing for co-expression of SY^WT or SY^R621C as indicated. The panel on the left represents the average S129-phosphorylation as determined by immunodetection using a P-S129 specific monoclonal antibody, shown in the right panel, and quantified relative to intensity obtained for immunodetection with a polyclonal α-Syn antibody. B: The left panel shows the average number of H4 neuroglioma cells containing inclusions formed by α-Syn–EGFP when expressed alone or in combination with SY^WT as determined by fluorescence microscopic visualization, for which a representative picture is shown in the right panel. C: Phosphorylation of α-Syn at S129 in H4 neuroglioma cells as detected by immunodetection using a P-S129 specific monoclonal antibody and quantified relative to the intensity obtained for immunodetection with a polyclonal α-Syn antibody. The panel on the left represents the relative average phosphorylation, the panel on the right a corresponding Western blot analysis. All data represent the mean ± SEM of at least three independent experiments. Significance was assayed using a 1-way ANOVA (A) or t-test (B and C)(* = p<0.05; ** = p<0.01; *** = p<0.001).</p

Sir2 mediates synphilin-1 toxicity in yeast.

<p>A: Relative quantification of viable cells as determined by their ability to form colonies at different times after inoculation of the wild-type strain or the isogenic <i>sir2Δ</i> mutant transformed with empty plasmids or constructs allowing for expression of α-Syn or SY^WT, either alone or in combination as indicated. The number of viable cells in samples taken after 24 h of growth of the two strains transformed with the empty plasmids was set at 100%. B and C: Quantification of viable cells (B) and cells producing ROS (C) during chronological ageing of the wild-type strain transformed with an empty plasmid (□) or expressing SY^WT (▪) and the isogenic <i>sir2Δ</i> mutant transformed with an empty plasmid (○) or expressing SY^WT (•). All data represent mean ± SEM of six independent transformants. Significance of the data was determined by t-tests (* = p<0.05; ** = p<0.01; *** = p<0.001).</p