Differential expression of mRNAs in a mouse model of pre-motor stage MSA.

<p>(A) Heatmaps represent significantly differentially expressed genes of RNA-seq (striatum, SN) and microarray (SN) analyses. For each gene (row), the log2-transformed change of the expression value in each sample to the average expression value over all samples is shown. Columns represent individual replicates grouped into MSA and control (WT) samples indicated by the blue (MSA) and grey (WT) bars at the top of the heatmaps. The color gradient indicates the expression change from negative to positive. The asterisks following gene names indicate overlapping genes between microarray and RNA-seq analyses in SN. (B) Venn diagram illustrating the number of overlapping differentially expressed mRNAs between SN and striatum tissue in MSA mice. (C) Heatmap highlights log₂-transformed fold changes of mRNAs overlapping between striatum und SN. Down-regulated mRNAs are indicated by a blue color gradient, whereas up-regulated miRNAs are indicated by an orange color gradient. mRNA with expression signals below background in the microarray experiment are highlighted in gray. From left to right, microarray and RNA-seq analysis results of SN and RNA-seq analysis of striatum are shown. Differential expression analysis of control versus transgenic MSA mice of both, striatum and SN samples, was performed by employing the DESeq2 package with predefined parameters [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.ref037" target="_blank">37</a>]. Genes with an adjusted p-value below 0.1 after multiple testing corrections were considered statistically significant [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.ref038" target="_blank">38</a>]. For microarray data differential gene expression was tested by a moderated t-test using the <i>limma</i> package [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.ref039" target="_blank">39</a>]. For both methods genes with an adjusted p-value < 0.1 after multiple testing corrections were considered statistically significant [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.ref038" target="_blank">38</a>].</p

Motor analysis of MSA versus age-matched control mice.

<p>Motor analysis of MSA versus age-matched control mice.</p

Neuropathological and behavioral characterization of a mouse model of a pre-motor stage of MSA.

<p>(A) Human α-synuclein overexpression in MSA transgenic mice resulted in α-synuclein accumulation in oligodendrocytes (arrows) detectable both in substantia nigra and striatum. (B) No dopaminergic neuronal loss was identified in the pre-motor stage in substantia nigra of MSA mice (n = 6) as compared to controls (n = 4) by stereological determination of the number of tyrosine hydroxylase (TH)-immunoreactrive (IR) neurons. (C) No GABAergic medium spiny neurons loss was identified in the pre-motor stage in striatum of MSA mice (n = 6) as compared to controls (n = 4) by stereological determination of the number of DARPP-32-IR neurons. (D) Iba-1-IR was used to determine the number and activation status of microglia (type A, B, C, and D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.ref029" target="_blank">29</a>]) in MSA (n = 3) and control mice (n = 3). No significant differences were detected between the groups with predominant representation of type A resting microglia in both substantia nigra and striatum. (E) GFAP-immunohistochemistry was used to determine the level of astroglial activation in MSA (n = 5) and control mice (n = 3) in substantia nigra and striatum. No significant differences were identified between the groups. Statistical analysis of the neuropathological data to compare control and transgenic MSA mice was done by t-test analysis with GraphPad Prism 5.03 software. Statistical significance was set at p<0.05. Data are presented as mean ± SEM. (F) TUNEL staining detected no cell death in SN and striatum of PM3 MSA mice. As a positive control we applied aged PM12 MSA mice (an age when detectable neuronal loss is recorded) that demonstrated positive TUNEL staining.</p

Differential expression of miRNAs in a mouse model of pre-motor stage MSA.

<p>(A) Heatmap shows expression changes of miRNAs of striatum (left) and SN (right). miRNAs with statistically significant (adjusted p<0.1) changes are indicated by a red line on the side. Gray boxes designate miRNAs with expression signals below background. The color gradient shows positive and negative log₂-transformed fold changes in orange and blue color, respectively. (B) Fold change and adjusted p-value of the miRNAs of the mir-467 family. (C) Venn diagram illustrates the overlap of differentially expressed miRNAs between SN and striatum in MSA mice. Differential expression analysis was performed by calculating a linear model for each miRNA according to the guidelines for simple dye swap experiments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.ref039" target="_blank">39</a>]. Duplicated spots were considered in the linear model fit. This model was then employed to obtain test statistics by the empirical Bayes method providing stable estimations for the sample variance of a small number of arrays [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.ref044" target="_blank">44</a>]. All differentially expressed miRNAs with an adjusted p-value < 0.1 after multiple testing corrections as proposed by Benjamini and Hochberg were considered statistically significant [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.ref038" target="_blank">38</a>].</p

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications - Fig 7

<p><b>Deregulated miRNA-mRNA regulatory network in the striatum of MSA mice in pre-motor stage of disease:</b> Modules “Protein handling” (A) and “Metabolism” (B). Differentially expressed miRNAs with predicted negatively correlated differentially expressed mRNA targets assigned to the indicated GO-terms (light blue rectangles) are visualized by employing Cytoscape (version 3.2.1). Round nodes designate mRNA and triangle nodes miRNA. Node size is proportional to its degree. Fold change (log₂ transformed) for each node is ranging from -0.75 (red) to 1 (green). The shade of blue color of the interaction arrows indicates the degree (range -1.00–0.00) of negative correlation between miRNA-mRNA target 3’ UTR interaction. Interaction arrow thickness is proportional to the number of algorithms predicting the miRNA-mRNA target 3’ UTR interaction, ranging from one to four.</p

Deregulated miRNA-mRNA regulatory network to “Immune system process” in MSA mice in disease pre-motor stage.

<p>Differentially expressed miRNAs with predicted negatively correlated differentially expressed mRNA targets are visualized by employing Cytoscape (version 3.2.1). Round nodes show mRNA and triangle nodes miRNA. Node size is proportional to its degree. Fold change (log₂ transformed) for each node is ranging from red (negative) to green (positive). Interaction arrow thickness is proportional to the number of algorithms predicting the miRNA-mRNA target 3’ UTR interaction, ranging from one to four. Differential expression of genes, in striatum and SN, such as <i>Anln</i>, <i>Car2</i>, <i>Cd59a</i>, <i>Hba-a1</i> and <i>Rps17</i>, is visualized by color corresponding to the mean fold change (exact values can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.s007" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150705#pone.0150705.s008" target="_blank">S3</a> Tables).</p