Isoquercitrin and localization of synaptotagmin-1.

<p><b>Left</b>, The immuno-staining images show that synaptotagmin-1 localizes especially at spots along the neurites induced by isoquercitrin (Q) alone or combined with Rho inhibitors (Y-27632, C3 transferase). Green = synaptotagmin-1, blue = nuclei. Scale bar = 50 micron. <b>Right</b>, The bar graph shows the quantification of the fluorescence spots along the neurites (n = 10 images, one-way ANOVA, * p<0.05). Data are shown as means ± s.e.m.</p

The Flavonoid Isoquercitrin Promotes Neurite Elongation by Reducing RhoA Activity

<div><h3>Background</h3><p>Neurite formation and synaptic patterning are fundamental to the development of a functional nervous system. Flavonoids are natural molecules known for having beneficial effects on brain health through diverse molecular pathways. Cytoskeletal changes occurring during neuritogenesis and synapse formation often involve Rho GTPases. Here we hypothesized that the flavonoid isoquercitrin promotes neuronal differentiation through Rho signalling.</p> <h3>Methodology/Principal Findings</h3><p>We performed time lapse imaging of NG108-15 cells during incubation with/without isoquercitrin. Isoquercitrin stimulated extensive neurites enriched in the synaptic vesicle protein synaptotagmin-1. Neurite extension was augmented by the ROCK inhibitor Y-27632 suggesting an inactivation of RhoA/Rho kinase as the mechanism. To test this, we first measured the dose-dependent effect of isoquercitrin on RhoA activity and found a 47% reduction in RhoA activity at concentrations which induced neurites (≥40 µM). Secondly, we tested the ability of isoquercitrin to rescue the neural phenotype in a model of RhoA-induced neurite retraction and found that 40 µM isoquercitrin added to cultures previously treated with the RhoA activator calpeptin produced significantly more neurite length/cell than calpeptin alone. Finally, we tested the hypothesis that isoquercitrin may affect RhoA localization preventing the translocation to the plasma membrane. Unexpectedly, immunolocalization studies showed that RhoA was present in nuclear compartments of control NG108-cells, but underwent translocation to the cytoplasm upon treatment with isoquercitrin. DNA microarrays and reverse transcription - quantitative PCR (RT-qPCR) revealed differences in global gene expression of Rho GTPase family members. These data taken together indicate that isoquercitrin is a potential stimulator of neuronal differentiation, through multiple Rho GTPase mediated mechanisms.</p> <h3>Conclusions/Significance</h3><p>As several members of the Rho GTPase family are implicated in human neurological disorders/injuries, our results suggest that isoquercitrin could be used in the treatment of these pathological states through its effect on this family of molecular switches.</p> </div

Isoquercitrin causes a change in RhoA localization.

<p><b>Upper left,</b> The fluorescent images (acquired with 63× oil objective) show immune-staining of RhoA in 48 hr cultures. Green = RhoA, blue = nuclei. Scale bar = 20 micron. <b>Bottom left,</b> 3D projections of z-stack images – obtained with Imaris software - corresponding to 2D images above. Scale bar = 15 micron. <b>Right,</b> The bar graph illustrates the standard deviation of the fluorescence in the nuclei normalized by the cytoplasmic fluorescence for each cell. (n = 10 images, pair sample t-Test, ****p<0.0001). Data are shown as means ± s.e.m.</p

Rho GTPase gene expression is modulated upon 24 hour isoquercitrin stimulation.

<p><i>rhoA</i> gene expression is down-regulated, while expression of <i>rhoQ</i> (TC10) is upregulated in the isoquercitrin treated cells in comparison with the control.</p

Time lapse acquisition of an isoquercitrin-stimulated NG108-15 cell.

<p>The images, acquired every 56 minutes, show the migration path and neurite extension out of the cell body. Note that the neurite tail undergoes a small amount of translation, where the top of each image is at the same position. The <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049979#pone.0049979.s006" target="_blank">video S1</a> is available in the Supplementary Information. Scale bar = 50 micron.</p

Isoquercitrin is able to restore the neural phenotype in calpeptin stimulated cells.

<p>The fluorescence images show 6 hr cultures stained with anti-neurofilament antibody. Green = neurofilament protein, blue = nuclei. Scale bar = 50 micron. <b>Bottom right</b>, The double y-axis graph shows: 1) the neurite length (micron)/cell (black bars) measured on the fluorescence images as described in M&M (n = 10 images, one-way ANOVA ****p<0.0001); 2) RhoA activation state (grey bars) evaluated through the Rho G LISA assay (n = 3, one-way ANOVA *p<0.05). Data are shown as means ± s.e.m.</p

Analysis of neurite length/cell vs. time.

<p><b>A,</b> Quantification of neurite length/cell upon addition of isoquercitrin alone or in combination with Rho inhibitors over a period of 48 hours (n = 3, one way ANOVA for repeated measures, F_3,8 = 17.363, P = 0.001). Acquisition started 30′ after the addition of the molecules. The maximum time for one imaging cycle was 12′. Post hoc Bonferroni test revealed significant differences between the control and the treatments of isoquercitrin combined with each Rho inhibitor, for alpha = 0.05. <b>B,</b> Decrease in cell proliferation, due to the presence of isoquercitrin (n = 3, pair-sample t-Test, *p<0.05). Data are shown as means ± s.e.m.</p

Calpeptin causes a rapid retraction of isoquercitrin induced neurites.

<p>After 48 hour incubation with isoquercitrin, neurite retraction was quantified upon addition of RhoA activator calpeptin over a period of 60 minutes (n = 3, one way ANOVA for repeated measures, F_1,4 = 27.310, P = 0.006). Acquisition was started 5′ after the addition of the molecule. The maximum time of one cycle of acquisition was about 1′. Post hoc Bonferroni test revealed significant differences between the control and the treatment with calpeptin, for alpha = 0.05. Data are shown as means ± s.e.m.</p

Isoquercitrin inhibits RhoA activity.

<p>Cells were maintained for 48 hours in the presence/absence of isoquercitrin and absorbance at OD_{490 nm} was calculated for the cell lysates according to Rho G-Lisa assay. <b>A,</b> Isoquercitrin dose dependent graph; <b>B,</b> Effects of isoquercitrin on RhoA activity alone and in combination with Rho/ROCK inhibitors (n = 3, one-way ANOVA, ** p<0.01; **** p<0.0001). Data are shown as means ± s.e.m.</p

Data analysis steps.

<p>(a) Data analysis pipeline. Alternative concepts: <i>(left column)</i> single parameter-based statistics, <i>(right column)</i> machine learning. (b) Spot detection for the EE assay. Spot intensities with the desired size <i>(black arrows)</i> were amplified, while noise <i>(red arrow)</i> and uneven background was suppressed. (c) Analysis of the EA and EF assays. GFP intensity was thresholded, and the detected objects were filtered by size. (d) (<i>Left)</i> EU assay confusion matrix. (<i>Right)</i> Comparison of classification methods using 10-fold cross validation. <i>Logistic regression classifiers with boosting</i> (LogitBoost) were the most accurate (∼98%) <i>(red arrow)</i>. (e) EI assay (<i>Left)</i> Original image. (<i>Middle)</i> Segmentation result. (<i>Right)</i> Phenotypic classification of cells: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068450#pone.0068450-Matlin1" target="_blank">[1]</a> import-negative <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068450#pone.0068450-deVries1" target="_blank">[2]</a> import-positive.</p