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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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Integrin αVβ3 expressed by DITNC1 astrocytes inhibits neurite extension of CAD cells.(A–D) Quantification of four different morphological parameters using IMARIS software (Bitplane, Switzerland) of bright-field microscopy images of CAD cells seeded over plastic, seeded over plastic in serum-free medium previously conditioned by DITNC1 cells for 24 hours (black bars) or over a monolayer of DITNC1 astrocytes in serum-free medium. (A) Percentage of differentiated CAD cells with processes ≥15 µm; (B) length of the processes extended by differentiated cells, expressed as a percentage of the control over plastic; (C) number of processes in 100 differentiated cells and (D) number of varicosities per 100 differentiated cells. (E,F) CAD cells seeded over fixed-astrocyte monolayers were induced to differentiate. To block αVβ3 integrin, fixed-cells were incubated with recombinant Thy-1-Fc or antibodies against β3 integrin. TRAIL-R2-Fc or antibodies against β1 integrin were used as controls. Co-cultures were photographed (E) and the percentage of differentiated CAD cells (F) was quantified as in (A). Arrows in E indicate axon-like neurites growing over the DITNC1 mololayer. All graphs show mean+s.e.m. determined from at least 100 cells per condition; n = 3. **P<0.01 or *P<0.05 compared with control cells seeded over plastic. #P<0.05 compared with their respective control.
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pone-0034295-g001: Integrin αVβ3 expressed by DITNC1 astrocytes inhibits neurite extension of CAD cells.(A–D) Quantification of four different morphological parameters using IMARIS software (Bitplane, Switzerland) of bright-field microscopy images of CAD cells seeded over plastic, seeded over plastic in serum-free medium previously conditioned by DITNC1 cells for 24 hours (black bars) or over a monolayer of DITNC1 astrocytes in serum-free medium. (A) Percentage of differentiated CAD cells with processes ≥15 µm; (B) length of the processes extended by differentiated cells, expressed as a percentage of the control over plastic; (C) number of processes in 100 differentiated cells and (D) number of varicosities per 100 differentiated cells. (E,F) CAD cells seeded over fixed-astrocyte monolayers were induced to differentiate. To block αVβ3 integrin, fixed-cells were incubated with recombinant Thy-1-Fc or antibodies against β3 integrin. TRAIL-R2-Fc or antibodies against β1 integrin were used as controls. Co-cultures were photographed (E) and the percentage of differentiated CAD cells (F) was quantified as in (A). Arrows in E indicate axon-like neurites growing over the DITNC1 mololayer. All graphs show mean+s.e.m. determined from at least 100 cells per condition; n = 3. **P<0.01 or *P<0.05 compared with control cells seeded over plastic. #P<0.05 compared with their respective control.

Mentions: The effect of αVβ3 integrin on neurite outgrowth was first examined in cell lines. CAD neuron-like cells were seeded over a monolayer of DITNC1 astrocytes and induced to differentiate by serum deprivation. When seeded on plastic (Fig. 1A), cellular processes of ≥15 µm in length (a sign of morphological differentiation together with length of processes, as well as number of both processes and varicosities) were observed for ∼80% of the cells. In contrast, only ∼35% of the cells developed such extended processes over a monolayer of astrocytes (Fig. 1A). Quantification of the length of processes showed that neurites were ∼55% shorter in CAD cells differentiated over DITNC1 astrocytes (Fig. 1B). Moreover, the number of both processes and varicosities, beadlike swellings of these processes, were decreased by ∼25% and ∼40%, respectively (Fig. 1C,D). Alternatively, serum-free conditioned media obtained from astrocyte cultures did not affect any of the parameters that account for morphological differentiation of CAD cells on plastic (Fig. 1A–D, black bars). Thus, cell-to-cell contact is required for DINCT1 astrocytes to restrict neurite extension of CAD cells.


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)

Integrin αVβ3 expressed by DITNC1 astrocytes inhibits neurite extension of CAD cells.(A–D) Quantification of four different morphological parameters using IMARIS software (Bitplane, Switzerland) of bright-field microscopy images of CAD cells seeded over plastic, seeded over plastic in serum-free medium previously conditioned by DITNC1 cells for 24 hours (black bars) or over a monolayer of DITNC1 astrocytes in serum-free medium. (A) Percentage of differentiated CAD cells with processes ≥15 µm; (B) length of the processes extended by differentiated cells, expressed as a percentage of the control over plastic; (C) number of processes in 100 differentiated cells and (D) number of varicosities per 100 differentiated cells. (E,F) CAD cells seeded over fixed-astrocyte monolayers were induced to differentiate. To block αVβ3 integrin, fixed-cells were incubated with recombinant Thy-1-Fc or antibodies against β3 integrin. TRAIL-R2-Fc or antibodies against β1 integrin were used as controls. Co-cultures were photographed (E) and the percentage of differentiated CAD cells (F) was quantified as in (A). Arrows in E indicate axon-like neurites growing over the DITNC1 mololayer. All graphs show mean+s.e.m. determined from at least 100 cells per condition; n = 3. **P<0.01 or *P<0.05 compared with control cells seeded over plastic. #P<0.05 compared with their respective control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316703&req=5

pone-0034295-g001: Integrin αVβ3 expressed by DITNC1 astrocytes inhibits neurite extension of CAD cells.(A–D) Quantification of four different morphological parameters using IMARIS software (Bitplane, Switzerland) of bright-field microscopy images of CAD cells seeded over plastic, seeded over plastic in serum-free medium previously conditioned by DITNC1 cells for 24 hours (black bars) or over a monolayer of DITNC1 astrocytes in serum-free medium. (A) Percentage of differentiated CAD cells with processes ≥15 µm; (B) length of the processes extended by differentiated cells, expressed as a percentage of the control over plastic; (C) number of processes in 100 differentiated cells and (D) number of varicosities per 100 differentiated cells. (E,F) CAD cells seeded over fixed-astrocyte monolayers were induced to differentiate. To block αVβ3 integrin, fixed-cells were incubated with recombinant Thy-1-Fc or antibodies against β3 integrin. TRAIL-R2-Fc or antibodies against β1 integrin were used as controls. Co-cultures were photographed (E) and the percentage of differentiated CAD cells (F) was quantified as in (A). Arrows in E indicate axon-like neurites growing over the DITNC1 mololayer. All graphs show mean+s.e.m. determined from at least 100 cells per condition; n = 3. **P<0.01 or *P<0.05 compared with control cells seeded over plastic. #P<0.05 compared with their respective control.
Mentions: The effect of αVβ3 integrin on neurite outgrowth was first examined in cell lines. CAD neuron-like cells were seeded over a monolayer of DITNC1 astrocytes and induced to differentiate by serum deprivation. When seeded on plastic (Fig. 1A), cellular processes of ≥15 µm in length (a sign of morphological differentiation together with length of processes, as well as number of both processes and varicosities) were observed for ∼80% of the cells. In contrast, only ∼35% of the cells developed such extended processes over a monolayer of astrocytes (Fig. 1A). Quantification of the length of processes showed that neurites were ∼55% shorter in CAD cells differentiated over DITNC1 astrocytes (Fig. 1B). Moreover, the number of both processes and varicosities, beadlike swellings of these processes, were decreased by ∼25% and ∼40%, respectively (Fig. 1C,D). Alternatively, serum-free conditioned media obtained from astrocyte cultures did not affect any of the parameters that account for morphological differentiation of CAD cells on plastic (Fig. 1A–D, black bars). Thus, cell-to-cell contact is required for DINCT1 astrocytes to restrict neurite extension of CAD cells.

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