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Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1.

Herrera-Molina R, Frischknecht R, Maldonado H, Seidenbecher CI, Gundelfinger ED, Hetz C, Aylwin Mde L, Schneider P, Quest AF, Leyton L - PLoS ONE (2012)

Bottom Line: In differentiating primary neurons exposed to α(V)β(3)-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, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase.

View Article: PubMed Central - PubMed

Affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.

ABSTRACT
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 α(V)β(3) integrin in trans eliciting responses in astrocytes. Nonetheless, whether α(V)β(3) 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 α(V)β(3) integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous α(V)β(3) integrin restricted neurite outgrowth. Likewise, α(V)β(3)-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(-) CAD cells. In differentiating primary neurons exposed to α(V)β(3)-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, α(V)β(3)-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 α(V)β(3) integrin. Binding of α(V)β(3)-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that α(V)β(3) 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.

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Soluble αVβ3-Fc induces Thy-1 cluster formation on the plasma membrane of living cortical neurons.Mature cortical neurons were treated with supernatants containing αVβ3-Fc fusion protein (αVβ3-Fc) supplemented or not with Protein A (PA), or with αVβ3-Fc-depleted supernatants (DS) for 15–60 minutes. Neurons were immunostained for Thy-1 only (A) or for Thy-1 and αVβ3-Fc (D). (A and D) Thy-1 clusters (arrows in A) and co-localization with bound αVβ3-Fc (arrows in D) of representative images captured with an epifluorescence microscope are shown. Bar = 20 µm. (B) Data plotted as time versus the number of clusters were obtained from images processed using ImageJ. Ranges of cluster size from 20 to 100 pix2 (1 pix2 = 0.01 µm2). (C) Data plotted as a range of cluster sizes versus the number of clusters for each indicated condition. Results show mean+s.e.m. (12 neurons per condition, n = 6). **P<0.01 and ***P<0.001 compared with their respective control cells in DS at time 15 minutes. #P<0.05 between αVβ3-Fc-Protein A and αVβ3-Fc (D).
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pone-0034295-g007: Soluble αVβ3-Fc induces Thy-1 cluster formation on the plasma membrane of living cortical neurons.Mature cortical neurons were treated with supernatants containing αVβ3-Fc fusion protein (αVβ3-Fc) supplemented or not with Protein A (PA), or with αVβ3-Fc-depleted supernatants (DS) for 15–60 minutes. Neurons were immunostained for Thy-1 only (A) or for Thy-1 and αVβ3-Fc (D). (A and D) Thy-1 clusters (arrows in A) and co-localization with bound αVβ3-Fc (arrows in D) of representative images captured with an epifluorescence microscope are shown. Bar = 20 µm. (B) Data plotted as time versus the number of clusters were obtained from images processed using ImageJ. Ranges of cluster size from 20 to 100 pix2 (1 pix2 = 0.01 µm2). (C) Data plotted as a range of cluster sizes versus the number of clusters for each indicated condition. Results show mean+s.e.m. (12 neurons per condition, n = 6). **P<0.01 and ***P<0.001 compared with their respective control cells in DS at time 15 minutes. #P<0.05 between αVβ3-Fc-Protein A and αVβ3-Fc (D).

Mentions: Bearing in mind the previous results, the possibility that the αVβ3 integrin may induce clustering of its neuronal receptor Thy-1 was tested. For this purpose, mature neurons were exposed for short periods of time (15–60 minutes) to different supernatants and after fixation, cell surface Thy-1 was identified with antibodies under non-permeabilizing conditions (see Materials and Methods). In non-treated (not shown) or in control neurons treated with depleted supernatants (DS), numerous small clusters were detected on the cell surface (left panel, Fig. 7A). The number and size of these clusters increased significantly in the presence of αVβ3-Fc for 15 minutes (right panel, Fig. 7A). For processes of neurons exposed to αVβ3-Fc-depleted supernatants (DS), ∼450 clusters with an area of 20–100 pix2 were detected after 15–60 minutes (Fig. 7B). For the same area, the number of clusters increased to ∼950 in the presence of αVβ3-Fc after 15 minutes (Fig. 7B). The number of clusters decreased after longer treatments (compare αVβ3-Fc at 15 minutes with 30 and 60 minutes in Fig. 7B). Cumulative distribution of cluster sizes to analyze the frequency of appearance of different clusters was also estimated. However, no changes in cumulative distribution of cluster sizes were observed in neurons treated with αVβ3-Fc for 15 or 60 minutes (not shown) indicating that αVβ3-Fc increases the number of a wide-range of Thy-1 clusters. As expected, the number of clusters significantly increased upon incubation with Protein-A (Fig. 7C). Additionally, larger clusters (100–200 pix2) were observed in neurons incubated with αVβ3-Fc-Protein A (Fig. 7C), and the presence of αVβ3-Fc was detectable in such large Thy-1 clusters (Fig. 7D). Importantly, increased clustering correlated with the axonal terminal retraction found in αVβ3-Fc- and αVβ3-Fc-Protein A-treated neurons (Fig. 6). Thus, Thy-1 clustering is likely to represent the first event in the signaling cascade triggered by αVβ3 integrin in neurons.


Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1.

Herrera-Molina R, Frischknecht R, Maldonado H, Seidenbecher CI, Gundelfinger ED, Hetz C, Aylwin Mde L, Schneider P, Quest AF, Leyton L - PLoS ONE (2012)

Soluble αVβ3-Fc induces Thy-1 cluster formation on the plasma membrane of living cortical neurons.Mature cortical neurons were treated with supernatants containing αVβ3-Fc fusion protein (αVβ3-Fc) supplemented or not with Protein A (PA), or with αVβ3-Fc-depleted supernatants (DS) for 15–60 minutes. Neurons were immunostained for Thy-1 only (A) or for Thy-1 and αVβ3-Fc (D). (A and D) Thy-1 clusters (arrows in A) and co-localization with bound αVβ3-Fc (arrows in D) of representative images captured with an epifluorescence microscope are shown. Bar = 20 µm. (B) Data plotted as time versus the number of clusters were obtained from images processed using ImageJ. Ranges of cluster size from 20 to 100 pix2 (1 pix2 = 0.01 µm2). (C) Data plotted as a range of cluster sizes versus the number of clusters for each indicated condition. Results show mean+s.e.m. (12 neurons per condition, n = 6). **P<0.01 and ***P<0.001 compared with their respective control cells in DS at time 15 minutes. #P<0.05 between αVβ3-Fc-Protein A and αVβ3-Fc (D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316703&req=5

pone-0034295-g007: Soluble αVβ3-Fc induces Thy-1 cluster formation on the plasma membrane of living cortical neurons.Mature cortical neurons were treated with supernatants containing αVβ3-Fc fusion protein (αVβ3-Fc) supplemented or not with Protein A (PA), or with αVβ3-Fc-depleted supernatants (DS) for 15–60 minutes. Neurons were immunostained for Thy-1 only (A) or for Thy-1 and αVβ3-Fc (D). (A and D) Thy-1 clusters (arrows in A) and co-localization with bound αVβ3-Fc (arrows in D) of representative images captured with an epifluorescence microscope are shown. Bar = 20 µm. (B) Data plotted as time versus the number of clusters were obtained from images processed using ImageJ. Ranges of cluster size from 20 to 100 pix2 (1 pix2 = 0.01 µm2). (C) Data plotted as a range of cluster sizes versus the number of clusters for each indicated condition. Results show mean+s.e.m. (12 neurons per condition, n = 6). **P<0.01 and ***P<0.001 compared with their respective control cells in DS at time 15 minutes. #P<0.05 between αVβ3-Fc-Protein A and αVβ3-Fc (D).
Mentions: Bearing in mind the previous results, the possibility that the αVβ3 integrin may induce clustering of its neuronal receptor Thy-1 was tested. For this purpose, mature neurons were exposed for short periods of time (15–60 minutes) to different supernatants and after fixation, cell surface Thy-1 was identified with antibodies under non-permeabilizing conditions (see Materials and Methods). In non-treated (not shown) or in control neurons treated with depleted supernatants (DS), numerous small clusters were detected on the cell surface (left panel, Fig. 7A). The number and size of these clusters increased significantly in the presence of αVβ3-Fc for 15 minutes (right panel, Fig. 7A). For processes of neurons exposed to αVβ3-Fc-depleted supernatants (DS), ∼450 clusters with an area of 20–100 pix2 were detected after 15–60 minutes (Fig. 7B). For the same area, the number of clusters increased to ∼950 in the presence of αVβ3-Fc after 15 minutes (Fig. 7B). The number of clusters decreased after longer treatments (compare αVβ3-Fc at 15 minutes with 30 and 60 minutes in Fig. 7B). Cumulative distribution of cluster sizes to analyze the frequency of appearance of different clusters was also estimated. However, no changes in cumulative distribution of cluster sizes were observed in neurons treated with αVβ3-Fc for 15 or 60 minutes (not shown) indicating that αVβ3-Fc increases the number of a wide-range of Thy-1 clusters. As expected, the number of clusters significantly increased upon incubation with Protein-A (Fig. 7C). Additionally, larger clusters (100–200 pix2) were observed in neurons incubated with αVβ3-Fc-Protein A (Fig. 7C), and the presence of αVβ3-Fc was detectable in such large Thy-1 clusters (Fig. 7D). Importantly, increased clustering correlated with the axonal terminal retraction found in αVβ3-Fc- and αVβ3-Fc-Protein A-treated neurons (Fig. 6). Thus, Thy-1 clustering is likely to represent the first event in the signaling cascade triggered by αVβ3 integrin in neurons.

Bottom Line: In differentiating primary neurons exposed to α(V)β(3)-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, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase.

View Article: PubMed Central - PubMed

Affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.

ABSTRACT
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 α(V)β(3) integrin in trans eliciting responses in astrocytes. Nonetheless, whether α(V)β(3) 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 α(V)β(3) integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous α(V)β(3) integrin restricted neurite outgrowth. Likewise, α(V)β(3)-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(-) CAD cells. In differentiating primary neurons exposed to α(V)β(3)-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, α(V)β(3)-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 α(V)β(3) integrin. Binding of α(V)β(3)-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that α(V)β(3) 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.

Show MeSH
Related in: MedlinePlus