Additional file 2: Figure S2. of Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies

Characterisation of Tau 1N4R fibrillar assemblies. (a) Representative negatively stained TEM of sonicated fibrils. Scale bar, 100 nm. (b) Length distribution of sonicated Tau fibrils obtained by measuring the length of 227 fibrils in negatively stained TEM samples. (TIF 19797 kb

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

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

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

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 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

Analysis of Tau aggregation by electronic microscopy and Gold-immunolabeling.

<p>Lysates from differentiated Mock and 4RTau cell lines, treated with tetracycline for 48 hours, were subjected to immunogold labelling with TauCter 1902 (Total Tau) antibody and revealed by colloidal gold labelled secondary antibody. Images were obtained at high magnification: 20 000×. “Clustering” of Tau immunoreactivity is indicated by black arrows.</p

Analysis of Tau phosphorylation in 4RTau cell lines by 2D electrophoresis.

<p>A) Bi-dimensional analysis of <i>WT</i> and Mutated <i>P301S</i> or <i>S305N</i> 4RTau. Differentiated cells treated with tetracycline for 48 hours were subjected to 2D analysis and immunoblotted with Tau Exon10 antibody (4RTau specific). Alignments of immunoblots were performed using unspecific products revealed by secondary antibody. Different molecular weight (MW) levels (levels1–3: L1–L3) of Tau isovariants are represented by short dashed lines and pI indicators (obtained with an internal IEF control) by long and short mixed dashed lines. B) Bi-dimensional analysis of <i>WT</i> and Mutated 4RTau after <i>in vitro</i> Tau dephosphorylation followed by immunoblotting with Tau exon10 antibody. C) Immunoblot analysis of <i>in vitro</i> dephosphorylated <i>WT</i> and mutated (<i>P301S</i> and <i>S305N</i>) 4RTau. Differentiated cells treated with tetracycline for 48 hours were subjected (+) or not (−), to an <i>in vitro</i> dephosphorylation by λ-phosphatase and immunoblotted by phosphorylation dependent antibody. Total amount of Tau proteins loaded is visualized with TauCter 1902 antibody (Total Tau) and β-actin was used as loading control.</p