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BMP signaling in astrocytes downregulates EGFR to modulate survival and maturation.

Scholze AR, Foo LC, Mulinyawe S, Barres BA - PLoS ONE (2014)

Bottom Line: We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway.In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR).In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed.

View Article: PubMed Central - PubMed

Affiliation: Stanford University School of Medicine, Department of Neurobiology, Stanford, California, United States of America.

ABSTRACT
Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.

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BMP signaling modulates astrocyte maturation invitro.A–C. Purified astrocytes were cultured for 3DIV in HbEGF (A), BMP5 (B), or FGF2 and GFAP expression examined by immunostaining. CellMask HCS staining was used to visualize all astrocytes. Images show individual channels from the same field. Scale bars are 100 µm. The percentage of astrocytes expressing GFAP at 3DIV was quantified using ImageJ (C). D–G. Purified astrocytes were cultured for 3DIV in HbEGF or BMP5 and the expression of AQP4 and S100ß examined by immunostaining. All cultures were also stained with GFAP. Scale bars are 50 µm. H–J. The proliferative capabilities of purified astrocytes in HbEGF, BMP5 and FGF2 were quantified invitro over 7 days using clonal analysis. Average clone size was calculated for both individual growth factors (H) and combinations of factors (I). Clonal analysis was also performed on astrocytes pre-treated with BMP5 for 7DIV to examine reversibility (J). K–L. Western blots for EGFR. Protein samples collected from acutely purified astrocytes at different postnatal time points confirm strong developmental downregulation of astrocyte EGFR (K). EGFR is also strongly downregulated in astrocytes cultured with BMP5 for 6DIV, even in the presence of other growth factors (L). Actin bands confirm equal protein loading. N ≥3 for each condition. Significance determined using one-way ANOVA with Tukey correction. Error bars represent SEM.
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pone-0110668-g003: BMP signaling modulates astrocyte maturation invitro.A–C. Purified astrocytes were cultured for 3DIV in HbEGF (A), BMP5 (B), or FGF2 and GFAP expression examined by immunostaining. CellMask HCS staining was used to visualize all astrocytes. Images show individual channels from the same field. Scale bars are 100 µm. The percentage of astrocytes expressing GFAP at 3DIV was quantified using ImageJ (C). D–G. Purified astrocytes were cultured for 3DIV in HbEGF or BMP5 and the expression of AQP4 and S100ß examined by immunostaining. All cultures were also stained with GFAP. Scale bars are 50 µm. H–J. The proliferative capabilities of purified astrocytes in HbEGF, BMP5 and FGF2 were quantified invitro over 7 days using clonal analysis. Average clone size was calculated for both individual growth factors (H) and combinations of factors (I). Clonal analysis was also performed on astrocytes pre-treated with BMP5 for 7DIV to examine reversibility (J). K–L. Western blots for EGFR. Protein samples collected from acutely purified astrocytes at different postnatal time points confirm strong developmental downregulation of astrocyte EGFR (K). EGFR is also strongly downregulated in astrocytes cultured with BMP5 for 6DIV, even in the presence of other growth factors (L). Actin bands confirm equal protein loading. N ≥3 for each condition. Significance determined using one-way ANOVA with Tukey correction. Error bars represent SEM.

Mentions: The intermediate filament glial fibrillary acidic protein (GFAP) is a classic astrocyte marker that is expressed at different levels by mature astrocytes in different brain regions [23]. Astrocyte GFAP expression also varies across development and after injury [24]. Previous work has identified a link between BMP stimulation of NPCs and increased GFAP expression in the derived astrocyte lineage cells [19], [25], [26]. To quantify the direct effects of BMP signaling on GFAP expression in immature astrocytes, we employed immunostaining. Culture in BMP5 significantly increased the intensity of GFAP staining in astrocytes, as well as the fraction that expressed GFAP at 3DIV (56.9±2.6%, p<0.0001) as compared to either HbEGF (13.1±2.3%) or FGF2 (9.9±1.0%) (Fig. 3A–C). Thus, BMP signaling can directly increase GFAP expression in immature astrocytes, although our understanding of what variation in GFAP expression means for astrocyte maturation and function is still fairly limited.


BMP signaling in astrocytes downregulates EGFR to modulate survival and maturation.

Scholze AR, Foo LC, Mulinyawe S, Barres BA - PLoS ONE (2014)

BMP signaling modulates astrocyte maturation invitro.A–C. Purified astrocytes were cultured for 3DIV in HbEGF (A), BMP5 (B), or FGF2 and GFAP expression examined by immunostaining. CellMask HCS staining was used to visualize all astrocytes. Images show individual channels from the same field. Scale bars are 100 µm. The percentage of astrocytes expressing GFAP at 3DIV was quantified using ImageJ (C). D–G. Purified astrocytes were cultured for 3DIV in HbEGF or BMP5 and the expression of AQP4 and S100ß examined by immunostaining. All cultures were also stained with GFAP. Scale bars are 50 µm. H–J. The proliferative capabilities of purified astrocytes in HbEGF, BMP5 and FGF2 were quantified invitro over 7 days using clonal analysis. Average clone size was calculated for both individual growth factors (H) and combinations of factors (I). Clonal analysis was also performed on astrocytes pre-treated with BMP5 for 7DIV to examine reversibility (J). K–L. Western blots for EGFR. Protein samples collected from acutely purified astrocytes at different postnatal time points confirm strong developmental downregulation of astrocyte EGFR (K). EGFR is also strongly downregulated in astrocytes cultured with BMP5 for 6DIV, even in the presence of other growth factors (L). Actin bands confirm equal protein loading. N ≥3 for each condition. Significance determined using one-way ANOVA with Tukey correction. Error bars represent SEM.
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pone-0110668-g003: BMP signaling modulates astrocyte maturation invitro.A–C. Purified astrocytes were cultured for 3DIV in HbEGF (A), BMP5 (B), or FGF2 and GFAP expression examined by immunostaining. CellMask HCS staining was used to visualize all astrocytes. Images show individual channels from the same field. Scale bars are 100 µm. The percentage of astrocytes expressing GFAP at 3DIV was quantified using ImageJ (C). D–G. Purified astrocytes were cultured for 3DIV in HbEGF or BMP5 and the expression of AQP4 and S100ß examined by immunostaining. All cultures were also stained with GFAP. Scale bars are 50 µm. H–J. The proliferative capabilities of purified astrocytes in HbEGF, BMP5 and FGF2 were quantified invitro over 7 days using clonal analysis. Average clone size was calculated for both individual growth factors (H) and combinations of factors (I). Clonal analysis was also performed on astrocytes pre-treated with BMP5 for 7DIV to examine reversibility (J). K–L. Western blots for EGFR. Protein samples collected from acutely purified astrocytes at different postnatal time points confirm strong developmental downregulation of astrocyte EGFR (K). EGFR is also strongly downregulated in astrocytes cultured with BMP5 for 6DIV, even in the presence of other growth factors (L). Actin bands confirm equal protein loading. N ≥3 for each condition. Significance determined using one-way ANOVA with Tukey correction. Error bars represent SEM.
Mentions: The intermediate filament glial fibrillary acidic protein (GFAP) is a classic astrocyte marker that is expressed at different levels by mature astrocytes in different brain regions [23]. Astrocyte GFAP expression also varies across development and after injury [24]. Previous work has identified a link between BMP stimulation of NPCs and increased GFAP expression in the derived astrocyte lineage cells [19], [25], [26]. To quantify the direct effects of BMP signaling on GFAP expression in immature astrocytes, we employed immunostaining. Culture in BMP5 significantly increased the intensity of GFAP staining in astrocytes, as well as the fraction that expressed GFAP at 3DIV (56.9±2.6%, p<0.0001) as compared to either HbEGF (13.1±2.3%) or FGF2 (9.9±1.0%) (Fig. 3A–C). Thus, BMP signaling can directly increase GFAP expression in immature astrocytes, although our understanding of what variation in GFAP expression means for astrocyte maturation and function is still fairly limited.

Bottom Line: We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway.In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR).In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed.

View Article: PubMed Central - PubMed

Affiliation: Stanford University School of Medicine, Department of Neurobiology, Stanford, California, United States of America.

ABSTRACT
Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.

Show MeSH