Ezrin Mediates Neuritogenesis via Down-Regulation of RhoA Activity in Cultured Cortical Neurons

<div><p>Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (<i>Vil2^kd/kd</i>) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (<i>Vil2^+/+</i>) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the <i>Vil2^kd/kd</i> mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the <i>Vil2^kd/kd</i> neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the <i>Vil2^kd/kd</i> neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.</p></div

Increased RhoA activity in the Vil2^kd/kd neurons.

<p><i>A-C</i>, The amounts of active and total RhoA (<i>A</i>), Rac1 (<i>B</i>) and Cdc42 (<i>C</i>) from cell lysates of the <i>Vil2^+/+</i> and <i>Vil2^kd/kd</i> neurons (2 DIV). Representative patterns were presented. <i>D-F</i>, The ratios of active RhoA (<i>D</i>), Rac1 (<i>E</i>) and Cdc42 (<i>F</i>) to total amount of proteins were compared between the <i>Vil2^+/+</i> (white columns) and <i>Vil2^kd/kd</i> (black columns) neurons. Each experiment was performed in triplicate. <i>*p</i><0.05 , Student's t test. Data represent mean ± SE.</p

Neuritogenesis is impaired by ezrin knockdown.

<p><i>A</i>, <i>B</i>, The <i>Vil2^+/+</i> (<i>A</i>) and <i>Vil2^kd/kd</i> (<i>B</i>) neurons were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm. <i>C</i>, Stacked bar graph showing stage progression in the <i>Vil2^+/+</i> (n = 153) and <i>Vil2^kd/kd</i> (n = 162) neurons. Stage of cells were defined by the length of the longest neurite as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105435#pone.0105435-Hirai1" target="_blank">[19]</a>. <i>D-F</i>, Quantitation of number (<i>D</i>) and length (<i>E</i>) of neurites, and length of axon (<i>F</i>) in the <i>Vil2^+/+</i> (gray columns, n = 50) and <i>Vil2^kd/kd</i> (green columns, n = 50) neurons. Three independent experiments were performed. <i>*p</i><0.05, <i>**p</i><0.01, <i>***p</i><0.001, Student's t test. Data represent mean ± SE.</p

Y-27632 rescues neuritogenesis.

<p><i>A-D</i>, The <i>Vil2^+/+</i> and <i>Vil2^kd/kd</i> neurons treated with DMSO (<i>A</i>,<i>C</i>) or 40 µM Y-27632 (24 h, <i>B</i>,<i>D</i>) were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm. <i>E-G</i>, The number (<i>E</i>) and length (<i>F</i>) of neurites, and length of axon (<i>G</i>) were quantified in the <i>Vil2^+/+</i> and <i>Vil2^kd/kd</i> neurons treated with DMSO (white columns, n = 30) or 40 µM Y-27632 (black columns, n = 30). Three independent experiments were performed. <i>*p</i><0.05, <i>**p</i><0.01, <i>***p</i><0.001 (DMSO-treated vs. Y-27632-treated), <i>###p</i><0.001 (DMSO-treated <i>Vil2^+/+</i> vs. DMSO-treated <i>Vil2^kd/kd</i>), Student's t test. Data represent mean ± SE.</p

Distribution of ezrin in wild-type cultured cortical neurons at the stages 1, 2 and 3 was observed by immunofluorescence.

<p>Neurons at the stages 1, 2 and 3 were stained with an anti-ezrin antibody, rhodamine phalloidin, and an anti-α-tubulin antibody, respectively. In the bottom lane, neurons were triple stained with an anti-ezrin antibody (green), rhodamine phalloidin (red) and an anti-α-tubulin antibody (blue). Scale bars, 50 µm.</p