Astrocytic αVβ3 Integrin Inhibits Neurite Outgrowth and Promotes Retraction of Neuronal Processes by Clustering Thy-1

Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to αVβ3 integrin in trans eliciting responses in astrocytes. Nonetheless, whether αVβ3 integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of αVβ3 integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous αVβ3 integrin restricted neurite outgrowth. Likewise, αVβ3-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(−) CAD cells. In differentiating primary neurons exposed to αVβ3-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, αVβ3-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by αVβ3 integrin. Binding of αVβ3-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, αVβ3-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that αVβ3 integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage

Simultaneous Single Unit Recording in the Mitral Cell Layer of the Rat Olfactory Bulb under Nasal and Tracheal Breathing

Odor perception depends on the odorant-evoked changes on Mitral/Tufted cell firing pattern within the olfactory bulb (OB). The OB exhibits a significant "ongoing" or spontaneous activity in the absence of sensory stimulation. We characterized this ongoing activity by simultaneously recording several single neurons in the mitral cell layer (MCL) of anesthetized rats and determined the extent of synchrony and oscillations under nasal and tracheal breathing. We recorded 115 neurons and found no significant differences in the mean firing rates between both breathing conditions. Surprisingly, nearly all single units exhibited a long refractory period averaging 14.4 ms during nasal respiration that was not different under tracheal breathing. We found a small incidence (2% of neurons) of gamma band oscillations and a low incidence (8.1%) of correlated firing between adjacent MCL cells. During nasal respiration, a significant oscillation at the respiratory rate was observed in 12% of cells that disappeared during tracheal breathing. Thus, in the absence of odorants, MCL cells exhibit a long refractory period, probably reflecting the intrinsic OB network properties. Furthermore, in the absence of sensory stimulation, MCL cell discharge does not oscillate in the gamma band and the respiratory cycle can modulate the firing of these cell

Recombinant α_Vβ₃-Fc induces Thy-1 redistribution and process retraction in differentiated CAD cells.

<p>CAD cells were seeded on coverslips in complete media for 12 hours. Differentiation was induced or not (undifferentiated, <b>UD</b>), by changing to serum-free medium for 1, 24 or 72 hours. Then, cells were stained for Thy-1 (green, arrowheads) and polymerized actin (red). Samples were visualized by confocal microscopy. Images representative of those obtained in 5 independent experiments are shown. Magnification bars = 20 µm. (<b>B,C</b>) CAD cells differentiated for 24 hours were treated with supernatants containing (<b>α_Vβ₃-Fc</b>) or not (<b>control</b>) α<b>_V</b>β<b>₃</b>-Fc fusion protein. Cells were immunostained for Thy-1 (green) and polymerized actin (red). Fluorescence intensities of one (<b>B</b>) or 100 (<b>C</b>) neuronal-like processes were quantified by densitometric analysis using <i>ImageJ</i> software. Merged colors are shown in yellow (<b>B</b>). Average fluorescence intensities of the red (F-actin) and green (Thy-1) staining were obtained for control and α<b>_V</b>β<b>₃</b>-Fc treated cells (<b>C</b>). To compare several processes of different lengths, each fluorescence profile was fitted to a 0–100 scale (n = 5).</p

Time course of process retraction induced by recombinant α_Vβ₃-Fc.

<p>CAD cells were plated and differentiation was induced as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034295#pone-0034295-g004" target="_blank">Fig. 4<i>A</i></a> for 72 hours. Differentiated CAD cells were exposed to (<b>A</b>) supernatants depleted of recombinant α<b>_V</b>β<b>₃</b>-Fc (<b>Control</b>) or (<b>B</b>) supernatants containing α<b>_V</b>β<b>₃</b>-Fc for 75 minutes (<b>α_Vβ₃-Fc</b>). Cell morphology was continuously monitored using a digital camera coupled to a microscope with a water-immersion objective and DIC optics. Images captured every 2.5 minutes are shown. White arrowheads indicate the position of the tip of the neurite at time point zero, whereas the black arrowhead shows the end of the trajectory after 75 minutes in (<b>A</b>) or after 12.5, 27.5, 42.5, 57.5 and 75 minutes in (<b>B</b>). A representative result of 5 independent retraction experiments is shown.</p

Collapse of axonal terminals of cortical neurons induced by recombinant α_Vβ₃-Fc.

<p>(<b>A, B</b>) Differentiated cortical neurons, 12–15 days in culture <i>in vitro</i>, were treated with supernatants containing α<b>_V</b>β<b>₃</b>-Fc fusion protein (<b>α_Vβ₃-Fc</b>) or with α<b>_V</b>β<b>₃</b>-Fc-depleted supernatants (<b>DS</b>), supplemented or not with Protein A. Then, neurons were fixed and immunostained for Thy-1 (<b>A–D</b>) and Tau protein (<b>D</b>) and photographed using conventional fluorescence microscopy. (<b>B</b>) Digital zoom was applied to marked areas (red squares in A) and indicated areas were inverted by gray-tone scaling using <i>ImageJ</i>. (<b>C</b>) Quantification of Thy-1-positive retracted axonal terminals in neurons for each condition is plotted as a percentage of total cells. <b>NT</b>, non-treated neurons; <b>DS</b>, α<b>_V</b>β<b>₃</b>-Fc-depleted supernatants; <b>α_Vβ₃-Fc</b>, α<b>_V</b>β<b>₃</b>-Fc-containing supernatant; <b>PA</b>, Protein A; <b>α_Vβ₃-Fc+PA</b>, α<b>_V</b>β<b>₃</b>-Fc-containing supernatant supplemented with Protein A (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034295#s2" target="_blank">Methods</a>). Graphs show means+s.e.m. from at least 25 neurons per condition, n = 4. *, &, <i>P</i><0.05 compared to their respective control. (<b>D</b>) Representative axonal terminals are shown from neurons treated with α<b>_V</b>β<b>₃</b>-Fc-depleted supernatants (<b>DS</b>); α<b>_V</b>β<b>₃</b>-Fc-containing supernatant (<b>α_Vβ₃-Fc</b>); or 1 U/ml of PI-PLC added prior addition of α<b>_V</b>β<b>₃</b>-Fc-containing supernatants (<b>PI-PLC</b>/<b>α_Vβ₃-Fc</b>). Arrows point to Thy-1 clusters in axon and growth cone. Asterisk indicates a retracted axonal terminal.</p

Recombinant α_Vβ₃-Fc inhibits extension of dendrites in cortical neurons.

<p>(<b>A</b>) Cortical neurons, cultured for 4 and 7 days of culture <i>in vitro</i> were stained for Thy-1 (red), MAP-2 (soma/dendrite staining, blue) and Tau (axon staining, green). Arrows indicate some areas with Thy-1 staining. Asterisks label Thy-1-negative axons. (<b>B–E</b>) Neurons, cultured for 4–5 days <i>in vitro</i> were treated with supernatants containing α<b>_V</b>β<b>₃</b>-Fc fusion protein (<b>α_Vβ₃-Fc</b>), α<b>_V</b>β<b>₃</b>-Fc-depleted supernatants (<b>DS</b>), α<b>_V</b>β<b>₃</b>-Fc-depleted supernatants supplemented with TRAIL-R2-Fc (<b>TRAIL-R2-Fc</b>), or non-treated (<b>NT</b>) for 72 hours. Where indicated, 1 U/ml of PI-PLC was added prior to integrin addition (<b>D,E</b>). (<b>B,D</b>) Inverted fields of MAP-2 fluorescence images that were used to count neurons and evaluate dendrite length. (<b>C,E</b>) Quantification of two different morphological parameters performed using Neuro <i>ImageJ</i> software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034295#s3" target="_blank">Results</a> shown are the mean+s.e.m. of 720 neurons from three independent experiments (<b>C</b>) or 240 neurons from two independent experiments (<b>E</b>). *<i>P</i><0.05 compared with DS condition.</p