Essential node sub-network.

<p>Node essentiality is determined by considering nodes having both degree centrality and weighted betweenness centrality over the upper-quartile. Essential nodes are placed in non-redundant portions of the network and thus cannot be removed without a deep impact on network connectivity. These genes intercept the network backbone, represented by the axis <i>TCF7L2-JUN-MAK8-PRKACG</i>, carrying the top perturbation levels, especially in proximity of the sources. Nodes and edges are labelled according to the conventions followed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185797#pone.0185797.g002" target="_blank">Fig 2</a>.</p

Steiner tree.

<p>Steiner tree obtained applying a Shortest Path Heuristic (SPH) algorithm. The tree has 167 nodes and 166 edges. The size of each node (i.e. gene) is proportional to its degree (i.e. the number of incoming and outgoing connections). Node colours indicate: perturbed seeds (green), non-perturbed seeds (red), perturbed connectors (blue), and non-perturbed connectors (yellow). Red edges correspond to perturbed interactions, while edge thickness is proportional to their weight (i.e. their perturbance level). A perturbed interaction has a weight <i>w</i> < 0.33 (i.e. p < 0.05 threshold over the nominal p-value). The entire network is characterized by a backbone <i>CAMK2A-TCF7L2- CTNNB1-JUN-MAK8-PRKACG</i>, where <i>CAMK2A</i> and <i>MAPK8-PRKACG</i> are the main perturbed hubs, <i>CTNNB1</i> and <i>JUN</i> represent the sinks of the entire system, and <i>TCF7L2</i> is the bottleneck connecting them.</p

ncWNT sub-network.

<p>This sub-network focuses on the module characterized by a series of receptors and enzymes regulating Calcium/cAMP homeostasis and involved in the non-canonical Ca⁺²/WNT signaling pathway. Among them, the most perturbed are the <i>EGFR</i> receptor and its target phospholipase <i>PLCB3</i>, and the routes <i>ITPR1-CAMK2A-CALM2-PPP3CC</i> and <i>ITPR1-CAMK2A-EP300-TCF7L2</i>. <i>JUN</i> is a large sink between this module and the MAPK-JNK one. Nodes and edges are labelled according to the conventions followed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185797#pone.0185797.g002" target="_blank">Fig 2</a>.</p

Data analysis workflow.

<p>Our network-based data analysis method includes three inputs: (i) a reference interactome that is used as a gene-gene interaction space; (ii) a set of seed nodes representing terminals (sources and targets) of information spreading through the interactome; and (iii) quantitative data used to build network weights. Weights are then used to generate a Steiner tree connecting seed genes through paths maximizing edge perturbation, using a weighted heuristic shortest path algorithm. The resulting Steiner tree is then converted into a Structural Equation Model (SEM) and fitted, to assess its validity. During SEM-based procedure, covariance between pairs of leaf genes (i.e., ancestral bow-free nodes) are tested and fitted using a latent variable (LV) model. The group variable <i>C</i> = {0, 1} influences a LV, modelling the unobserved cause(s) acting on the two target genes. Significant covariances are retained in the extended network, representing the final disease-network.</p

SEM goodness of fit.

<p>Goodness of fit measures for different models, fitted to multivariate data of the extracted Steiner tree. The selected model, indicated by (*), has the lowest Akaike Information Criterion (AIC = -9324.98).</p

MAPK-JNK sub-network.

<p>This sub-network focuses on the module characterized by a series Serine/Threonine kinases involved in the MAPK-JNK signaling pathway. Among them, <i>MAPK8</i> is the one having the highest outgoing connectivity of the entire network, and the most perturber incoming interaction carried by <i>PRKACG</i>, another FTD-network hub. Other deeply perturbed interactions include <i>MAPK8-TNF</i>, <i>MAPK8-CRK</i>. <i>JUN</i> is a large sink within this module and the non-canonical WNT pathway (ncWNT) one. Nodes and edges are labelled according to the conventions followed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185797#pone.0185797.g002" target="_blank">Fig 2</a>.</p

Additional file 1: of Rac1 activation links tau hyperphosphorylation and Aβ dysmetabolism in Alzheimer’s disease

Figure S1. Rac1 mutant peptides have high penetration due to the TAT sequence. (A-C) Representative confocal images of cortical neurons treated at DIV3 with different concentrations of TAT-GFP: 5 μM (A), 10 μM (B, C). After treatment, cells were fixed and stained for visualization of dendrites (MAP2) and nuclei (DAPI). Confocal analysis showed that TAT-GFP was internalized (single plane), also in live cells directly imaged 1h after treatment. Scale bars 10 μm. (D) MTT assay on primary cortical neurons after 24h from the administration of 2 μM Rac1 mutant peptides. The cell viability is expressed as % as compared to control. The data represented are mean ±SEM of four independent experiments, each done in triplicate. Figure S2. Aβ1-42 administration does not interfere with Rac1 localization or activation. (A) MTT assay on primary cortical neurons after 24h Aβ1-42 treatment at the indicated concentrations The Aβ peptide suspension was incubated 12h at 4°C prior treatment. The cell viability is expressed as % as compared to control. The data represented are mean ±SEM of four independent experiments, each done in triplicate. One-sample t test to a hypothetical mean of 100% was performed. (B) Representative dot-blot analysis of Aβ1-42 preparations with 6E10 and A11 antibodies. The protein concentration was 0.12 μg for 6E10 and 0.72 μg for A11 (C) Representative confocal images of primary cortical neurons treated with 0.1 μM Aβ1-42 between DIV11 and DIV14. Cells were stained against Rac1-GTP, F-actin, and neurofilament. Scale bars 30 μm. Figure S3. Efficacy of the subcellular fractionation. Representative blots of the subcellular fractionation experiments showing the levels of GluR1, LaminB, and SET in the membrane and nuclear fractions of SH-SY5Y cells. Four independent samples were assessed for the 2 fractions. Figure S4. Tau induced hyperphosphorylation does not alter Rac1 levels or activation. (A) Representative confocal pictures of mature cortical neurons treated with 10nM OA for 6h and immunostained against pS262 tau. Scale bar 30 μm. (B-C) Tau pS262 phosphorylation was analysed by western blot after 3 and 6h from OA administration. The data represented are mean with SEM of four or six independent experiments (3h treatment n=6, 6h treatment n=4). (D-E) Rac1-GTP pull done assay was performed after 3 and 6h from OA administration. The data represented are mean with SEM of three independent experiments. ns, not significant. Asterisks indicate unspecific bands. (DOCX 3215 kb